From 033e92093215b95f0eb7e48222e424740a71bb89 Mon Sep 17 00:00:00 2001 From: Johan Dahlin Date: Sat, 22 Nov 2025 20:47:38 +0100 Subject: [PATCH] add some pdfs --- ...ering_Eukaryot_DNA replikation_VT2025-1.pdf | 3 + ...ch terminering av eukaryot replikation-5.pdf | 3 + ... Functions, Biosynthesis, and Processing.md | 2366 +++++++++++++++++ ...Functions, Biosynthesis, and Processing.pdf | 3 + .../Kromatin/Instuderingsfrågor_Kromatin-5.pdf | 3 + .../Kromatin/Kromatin_VT2025-1.pdf | 3 + ... 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Discuss the primary function of RNA polymerases, the reaction they catalyze, and + +the chemical mechanism of that reaction. +2. Describe transcription, including the processes of initiation, elongation, and + +termination. +3. Compare the roles of eukaryotic RNA polymerases I, II, and III in producing + +ribosomal, transfer, and messenger RNAs. +4. Recognize the significance of transcription factors and enhancers in the regulation + +of transcription in eukaryotes. +5. Describe the process of RNA splicing, including the roles of the spliceosome and + +self-splicing RNA molecules. +6. Understand some of the differences between transcription in bacteria and in + +eukaryotes. + + +DNA stores genetic information in a stable form that can be readily +replicated. The expression of this genetic information requires its +flow from DNA to RNA and, usually, to protein, as was introduced in + +. +Chapter 8 This chapter examines transcription, which, you will +recall, is the process of synthesizing an RNA transcript from a DNA +template, transferring the sequence information within the DNA to +the new RNA molecule. We begin with a brief discussion of the +diverse types of RNA molecules; then we will turn to RNA +polymerases, the large and complex enzymes that carry out the + + +synthetic process. This will lead into a discussion of transcription in +bacteria and focus on the three stages of transcription: promoter +binding and initiation, elongation of the nascent RNA transcript, +and termination. We then examine transcription in eukaryotes, +focusing on the distinctions between bacterial and eukaryotic +transcription. + + +# **29.1 RNA Molecules Play Different** **Roles, Primarily in Gene Expression** + +While the function of some RNAs has been known for some time, +other classes of RNAs have only recently been discovered. The +investigation of some of these RNA molecules has been one of the +most productive areas of biochemical research in recent years. + +### **RNAs play key roles in protein** **biosynthesis** + + +As we will explore in Chapter 30, the long-known ribosomal and +transfer RNA molecules, along with messenger RNAs, are central to +protein synthesis. Ribosomal RNAs are critical components of +ribosomes (the sites of protein synthesis), and transfer RNAs play a +role in delivering amino acids (the building blocks of proteins) to the +ribosome. Messenger RNAs carry the information that ribosomes +use for the production of specific protein sequences. + +### **Some RNAs can guide modifications of** **themselves or other RNAs** + + +In eukaryotes, one of the most striking examples of RNA +modification is the splicing of mRNA precursors, a process that is +catalyzed by large complexes composed of both proteins and small +nuclear RNAs. These small nuclear RNAs play a crucial role in +guiding the splicing of messenger RNAs. + + +Remarkably, some RNA molecules can splice themselves in the +absence of other proteins and RNAs. This landmark discovery of +self-splicing introns, which we will discuss later in this chapter, +revealed that RNA molecules can serve as catalysts, which greatly +influenced our view of molecular evolution. Many other types of +RNAs, such as small regulatory RNAs and long noncoding RNAs, +have been discovered more recently, and while their functions are +still under active investigation, our understanding is rapidly +expanding. + +### **Some viruses have RNA genomes** + + +While DNA is the genetic material in most organisms, some viruses +have genomes made of RNA (Section 8.4). RNA viruses are +responsible for several diseases, such as influenza, polio, mumps, +Ebola, the common cold, and — of particular note — COVID-19. The +coronavirus SARS-CoV-2, which causes COVID-19, belongs to a +family of coronaviruses that have an unusually large, singlestranded RNA genome. Coronaviruses cause a variety of diseases in +mammals and birds that have a wide range of symptoms, including +respiratory distress in humans that can potentially be lethal. + + +RNA viruses vary in their genome organization. They have either +single- or double-stranded RNA molecules arranged in single or +multiple fragments. Inside their hosts, RNA viruses replicate their +genomes with a virus-encoded RNA polymerase that uses RNA as a +template. In some viruses, a complementary RNA molecule is made +from the single-stranded viral RNA genome, while in other viruses, a +double-stranded DNA copy is made that can integrate itself into the +host’s genome. Viral RNA polymerases do not have the same +proofreading ability of other polymerases, which leads to high +mutation rates of RNA viruses. + + +### **Messenger RNA vaccines provide** **protection against diseases** + +Messenger RNA (mRNA) vaccines take advantage of the fact that +cells can be tricked into making proteins they don’t usually make, +even those from other organisms such as viruses. Unlike other +vaccines that use parts of a weakened or inactivated pathogen to +trigger an immune response, mRNA vaccines — including those +available for the SARS-CoV-2 virus — use sections of pathogenic +(usually viral) mRNA that have been generated in the laboratory. +When human cells are injected with this mRNA, they will produce +the corresponding protein from this set of instructions. This +“foreign” protein will be presented on the surface of the cells, +trigger an immune response, and provide some level of protection if +and when the actual pathogen invades. Even though research on +mRNA vaccines had been going on for many years, the COVID-19 +pandemic accelerated the development, approval, and distribution +of mRNA vaccines to the public. + +##### **Self–Check Question** + + +Compare the biological roles of RNA and DNA. What aspects of the structure and +chemistry of RNA make it so versatile? + + +## **29.2 RNA Polymerases Catalyze** **Transcription** + +**Transcription**, the synthesis of RNA molecules from a DNA template, is +catalyzed by large enzymes called **RNA polymerases** . The basic +biochemistry of RNA synthesis is shared by all organisms, a +commonality that has been beautifully illustrated by the threedimensional structures of representative RNA polymerases from +prokaryotes and eukaryotes ( **Figure 29.1** ). Despite substantial +differences in size and number of polypeptide subunits, the overall +structures of these enzymes are quite similar, revealing a common +evolutionary origin. + + +RNA polymerases are very large, complex enzymes. For example, the +core of the RNA polymerase of _E. coli_ consists of five kinds of subunits + + +with the composition ( **Table 29.1** ). A typical eukaryotic RNA + + +polymerase is larger and more complex, having 12 subunits and a total +molecular mass of more than 500 kDa. Despite this complexity, the +detailed structures of RNA polymerases have been determined by x-ray +crystallography in work pioneered by Roger Kornberg and Seth Darst. +The structures of many additional RNA polymerase complexes have +been determined by cryo-electron microscopy. + + +**TABLE 29.1 Subunits of RNA polymerase from** _**E. coli**_ + + +α _rpoA_ 2 37 + + +β _rpoB_ 1 151 + + +_rpoC_ 1 155 + + +ω _rpoZ_ 1 10 + + +_rpoD_ 1 70 + + +#### **RNA synthesis comprises three stages:** **initiation, elongation, and termination** + +RNA synthesis, like all biological polymerization reactions, takes place +in three stages: _initiation_, _elongation_, and _termination_ . RNA polymerases +perform multiple functions in this process: + + +or +1. They search DNA for initiation sites, also called _promoter sites_ + +simply **promoters** . For instance, _E. coli_ DNA has about 2000 + + +promoters in its genome. + + +2. They unwind a short stretch of double-helical DNA to produce + +single-stranded DNA templates from which the sequence of bases +can be easily read out. + + +3. They select the correct ribonucleoside triphosphate and catalyze + +the formation of a phosphodiester bond. This process is repeated +many times as the enzyme moves along the DNA template. RNA +polymerase is completely processive — a transcript is synthesized +from start to end by a single RNA polymerase molecule. + + +4. They detect termination signals that specify where a transcript + +ends. + + +5. Their activity is regulated by activator and repressor proteins that + +interact with the promoter and modulate the ability of the RNA +polymerase to initiate transcription. Gene expression is controlled +substantially at the level of transcription, as will be discussed in +detail in . +Chapter 31 + + +The chemistry of RNA synthesis is identical for all forms of RNA, +including messenger RNAs, transfer RNAs, ribosomal RNAs, and small +regulatory RNAs, so the basic steps just outlined apply to all forms. +Their synthetic processes differ mainly in regulation, the specific RNA +polymerase that creates them, and their posttranscriptional processing. + + +#### **RNA polymerases catalyze the formation of a** **phosphodiester bond** + +The fundamental reaction of RNA synthesis, like that of DNA synthesis, +is the formation of a phosphodiester bond. The -hydroxyl group of the + + + last nucleotide in the chain makes a nucleophilic attack on the α +phosphoryl group of the incoming nucleoside triphosphate, releasing a +pyrophosphate. + + +The catalytic sites of all RNA polymerases include two metal ions, +normally magnesium ions ( **Figure 29.2** ). One ion remains tightly bound +to the enzyme, whereas the other ion comes in with the nucleoside +triphosphate and leaves with the pyrophosphate. Three conserved +aspartate residues participate in binding these metal ions. Given the +recent appreciation of the role of a third metal ion in the active site of +DNA polymerases (see Section 28.1), it will be interesting to see if RNA +polymerases show similarities to that model. + + +The polymerization reactions that are catalyzed by both prokaryotic and +eukaryotic RNA polymerases take place within a complex in DNA +termed a **transcription bubble** ( **Figure 29.3** ). This complex consists of +double-stranded DNA that has been locally unwound in a region of +approximately 17 base pairs. The edges of the bases that normally take +part in Watson–Crick base pairs are exposed in the unwound region. We +will begin with a detailed examination of the elongation process, +including the role of the DNA template read by RNA polymerase and the +reactions catalyzed by the polymerase, before returning to the more +complex processes of initiation and termination. + + +#### **RNA chains are formed de novo and grow in** **the -to- direction** + +Let us begin our examination of transcription by considering the DNA +template. The first nucleotide (the start site) of a DNA sequence to be +transcribed is denoted as and the second one as ; the nucleotide + +preceding the start site is denoted as . These designations refer to the + + +coding strand of DNA. Recall that the sequence of the _template strand_ of +DNA is the complement of that of the RNA transcript ( **Figure 29.4** ). In +contrast, the _coding strand_ of DNA has the same sequence as that of the +RNA transcript except for thymine (T) in place of uracil (U). The coding + +strand is also known as the _sense_ _strand_, and the template strand as + + +the _antisense_ _strand_ . + + + + +In contrast with DNA synthesis, RNA synthesis can start de novo, +without the requirement for a primer. Most newly synthesized RNA +chains carry a highly distinctive tag on the end: the first base at that + + +end is either pppG or pppA. + + +The presence of the triphosphate moiety confirms that RNA synthesis +starts at the end. + + +The dinucleotide shown above is synthesized by RNA polymerase as +part of the complex process of initiation, which will be discussed later +in the chapter. After initiation takes place, RNA polymerase elongates +the nucleic acid chain as follows ( **Figure 29.5** ). + + +1. A ribonucleoside triphosphate binds in the active site of the RNA + +polymerase directly adjacent to the growing RNA chain, and it +forms a Watson–Crick base pair with the template strand. + + +2. The -hydroxyl group of the growing RNA chain, which is oriented + + + and activated by the tightly bound metal ion, attacks the α +phosphoryl group to form a new phosphodiester bond, displacing +pyrophosphate. + + +3. Next, the RNA–DNA hybrid must move relative to the polymerase to + + +bring the end of the newly added nucleotide into proper position + + +for the next nucleotide to be added. This translocation step does not +include breaking any bonds between base pairs and is reversible; +but, once it has taken place, the addition of the next nucleotide, +favored by the triphosphate cleavage and pyrophosphate release +and cleavage, drives the polymerization reaction forward. + + +The lengths of the RNA–DNA hybrid and of the unwound region of DNA +stay rather constant as RNA polymerase moves along the DNA template. +The length of the RNA–DNA hybrid is determined by a structure within +the enzyme that forces the RNA–DNA hybrid to separate, allowing the +RNA chain to exit from the enzyme and the DNA chain to rejoin its DNA +partner ( **Figure 29.6** ). + + +#### **RNA polymerases backtrack and correct** **errors** + +The RNA–DNA hybrid can also move in the direction opposite that of +elongation ( **Figure 29.7** ). This backtracking is energetically less +favorable than moving forward because it breaks the bonds between a +base pair. However, backtracking is very important for _proofreading_ . The +incorporation of an incorrect nucleotide introduces a non-Watson–Crick +base pair. In this case, breaking the bonds between this base pair and +backtracking is energetically less costly. + + + + + +After the polymerase has backtracked, the phosphodiester bond one +base pair before the one that has just formed is adjacent to the metal ion +in the active site. In this position, a hydrolysis reaction in which a water +molecule attacks the phosphate can result in the cleavage of the + + +phosphodiester bond and the release of a dinucleotide that includes the +incorrect nucleotide. + + +Studies of single molecules of RNA polymerase have confirmed that the +enzymes pause and backtrack to correct errors. Furthermore, these +proofreading activities are often enhanced by accessory proteins. + + +or +The final error rate of the order of one mistake per + + +nucleotides is higher than that for DNA replication, including all errorcorrecting mechanisms. The lower fidelity of RNA synthesis can be +tolerated because mistakes are not transmitted to progeny. For most +genes, many RNA transcripts are synthesized; a few defective +transcripts are unlikely to be harmful. + +###### **Self–Check Question** + + +List some ways in which RNA polymerases are similar to DNA polymerases, and how they are +different. + +#### **RNA polymerase binds to promoter sites on** **the DNA template in bacteria to initiate** **transcription** + + +While the elongation process is common to all organisms, the processes +of initiation and termination differ substantially in bacteria and +eukaryotes. We begin with a discussion of these processes in bacteria, +starting with initiation of transcription. + + +The bacterial RNA polymerase discussed earlier with the composition + + +is referred to as the . The inclusion of an additional +_core enzyme_ + + +subunit ( σ ) produces the _holoenzyme_ with composition . The **σ** + +**subunit** helps find the promoters, sites on DNA where transcription +begins. At these sites, the σ subunit participates in the initiation of RNA +synthesis and then dissociates from the rest of the enzyme. + + +Sequences upstream of the promoter site are important in determining +where transcription begins. A striking pattern is evident when the +sequences of bacterial promoters are compared: Two common motifs +are present on the upstream side of the transcription start site. They are + +known as the and the +_sequence_ _sequence_ because they are +centered at about 10 and 35 nucleotides upstream of the start site. The +region containing these sequences is called the _core promoter_ . The + +and sequences are each 6 bp long. Their **consensus sequences**, +deduced from analyses of many promoters ( **Figure 29.8** ), are: + + +Promoters differ markedly in their efficacy. Some genes are transcribed +frequently — as often as every 2 seconds in _E. coli_ . The promoters for +these genes are referred to as _strong promoters_ . In contrast, other genes +are transcribed much less frequently, about once in 10 minutes; the +promoters for these genes are _weak promoters_ . The and +regions of most strong promoters have sequences that correspond +closely to the consensus sequences, whereas weak promoters tend to +have multiple substitutions at these sites. Indeed, mutation of a single +base in either the sequence or the sequence can diminish +promoter activity. + + +The distance between these conserved sequences also is important; a +separation of 17 nucleotides is optimal. Thus, the efficiency or strength +of a promoter sequence serves to regulate transcription. Regulatory +proteins that bind to specific sequences near promoter sites and +interact with RNA polymerase (Chapter 31) also markedly influence the +frequency of transcription of many genes. + + +Outside the core promoter in a subset of highly expressed genes is the +_upstream element_ (also called the UP element). This sequence is present +from 40 to 60 nucleotides upstream of the transcription start site. The +UP element is bound by the α subunit of RNA polymerase and serves to +increase the efficiency of transcription by creating an additional +interaction site for the polymerase. + +#### **Sigma subunits of RNA polymerase in** **bacteria recognize promoter sites** + + +To initiate transcription, the core of RNA polymerase must bind + + +the promoter. However, it is the σ subunit that makes this binding +possible by enabling RNA polymerase to recognize promoter sites. In +the presence of the σ subunit, the RNA polymerase binds weakly to the +DNA and slides along the double helix until it dissociates or encounters +a promoter. The σ subunit recognizes the promoter through several +interactions with the nucleotide bases of the promoter DNA. The +structure of a bacterial RNA polymerase holoenzyme bound to a +promoter site shows the σ subunit interacting with DNA at the and + + +regions essential to promoter recognition ( **Figure 29.9** ). Therefore, + +the σ +subunit is responsible for the specific binding of the RNA +polymerase to a promoter site on the template DNA. The σ subunit is +generally released when the nascent RNA chain reaches 9 or 10 +nucleotides in length. After its release, it can associate with another +core enzyme and assist in a new round of initiation. + + +#### **The template double helix must be unwound** **for transcription to take place** + +Although the bacterial RNA polymerase can search for promoter sites +when bound to double-helical DNA, a segment of the DNA double helix +must be unwound before synthesis can begin. The transition from the +closed promoter complex (in which DNA is double helical) to the open +promoter complex (in which a DNA segment is unwound) is an essential +event in both bacterial and eukaryotic transcription. In bacteria it is the +RNA polymerase itself that accomplishes this ( **Figure 29.10** ), while in +eukaryotes additional proteins are required to unwind the DNA +template. + + + + + +We know that, in bacteria, the free energy necessary to break the bonds +between approximately 17 base pairs in the double helix is derived from +additional interactions between the template and the bacterial RNA +polymerase. These interactions become possible when the DNA distorts +to wrap around the RNA polymerase; they also occur between the +single-stranded DNA regions and other parts of the enzyme. These +interactions stabilize the open promoter complex and help pull the +template strand into the active site. The element remains in a + + +double-helical state, whereas the element is unwound. The stage is + + +now set for the formation of the first phosphodiester bond of the new +RNA chain. + +#### **Elongation takes place at transcription** **bubbles that move along the DNA template** + + +The elongation phase of RNA synthesis begins with the formation of the +first phosphodiester bond, after which repeated cycles of nucleotide +addition can take place. However, until about 10 nucleotides have been +added, RNA polymerase sometimes releases the short RNA, which +dissociates from the DNA and gets degraded. Once RNA polymerase +passes this point, the enzyme stays bound to its template until a +termination signal is reached. + + +The region containing the unwound DNA template and nascent RNA +corresponds to the transcription bubble ( **Figure 29.11** ). The newly +synthesized RNA forms a hybrid helix with the template DNA strand. +This RNA–DNA helix is about 8 bp long, which corresponds to nearly + + +one turn of a double helix. The -hydroxyl group of the RNA in this + + +hybrid helix is positioned so that it can attack the α -phosphorus atom of +an incoming ribonucleoside triphosphate. The core bacterial RNA +polymerase also contains a binding site for the coding strand of DNA. + + +As in the initiation phase, about 17 bp of DNA are unwound throughout +the elongation phase. The transcription bubble moves a distance of 170 +Å (17 nm) in a second, which corresponds to a rate of elongation of +about 50 nucleotides per second. Although rapid, it is much slower than +the rate of DNA synthesis, which is 800 nucleotides per second. + +#### **Sequences within the newly transcribed RNA** **signal termination** + + +How does the RNA polymerase know where to stop transcription? In the +termination phase of transcription, the formation of phosphodiester +bonds ceases, the RNA–DNA hybrid dissociates, the unwound region of +DNA rewinds, and RNA polymerase releases the DNA. This process is as +precisely controlled as initiation. So what determines where + + +transcription is terminated? In both eukaryotes and bacteria, the +transcribed regions of DNA templates contain so-called _intrinsic_ +termination signals _._ + + +In bacteria, the simplest intrinsic termination signal is a palindromic +GC-rich region followed by an AT-rich region. The RNA transcript of this +DNA palindrome is self-complementary ( **Figure 29.12** ). Hence, its bases +can pair to form a hairpin structure with a stem and loop, a structure +favored by its high content of G and C residues. Guanine–cytosine base +pairs are more stable than adenine–thymine pairs, primarily because of +the preferred base-stacking interactions in G–C base pairs (Section 1.3). +This stable hairpin is followed by a sequence of four or more uracil +residues, which also are crucial for termination. The RNA transcript +ends within or just after them. + + +How does this combination hairpin–oligo(U) structure terminate +transcription? First, RNA polymerase likely pauses immediately after it +has synthesized a stretch of RNA that folds into a hairpin. Furthermore, +the RNA–DNA hybrid helix produced after the hairpin is unstable +because its rU–dA base pairs are the weakest of the four kinds. Hence, +the pause in transcription caused by the hairpin permits the weakly +bound nascent RNA to dissociate from the DNA template and then from +the enzyme. The solitary DNA template strand rejoins its partner to reform the DNA duplex, and the transcription bubble closes. + +#### **In bacteria, the rho protein helps to** **terminate the transcription of some genes** + + +Bacterial RNA polymerase needs no help to terminate transcription at +the intrinsic sites described above. At other sites, however, termination +requires the participation of an additional factor. This discovery was +prompted by the observation that some RNA molecules synthesized in +vitro by RNA polymerase acting alone are longer than those made in +vivo. The missing factor, a protein that caused the correct termination, +was isolated and named **rho (** **ρ** **)** . + + +Additional information about the action of ρ was obtained by adding +this termination factor to an incubation mixture at various times after +the initiation of RNA synthesis ( **Figure 29.13** ). RNAs with sedimentation +coefficients of 10S, 13S, and 17S were obtained when ρ was added at +initiation, a few seconds after initiation, and 2 minutes after initiation, +respectively. If no ρ was added, transcription yielded a 23S RNA product. +It is evident that the template contains at least three termination sites +that respond to ρ (yielding 10S, 13S, and 17S RNA) and one termination +site that does not (yielding 23S RNA). Thus, specific termination at a site +producing 23S RNA can take place in the absence of ρ . However, ρ +detects additional termination signals that are not recognized by RNA +polymerase alone. + + +The ρ protein promotes about 20% of termination events in bacteria, +but exactly how it selects its target termination signals is not clear. +Unlike the hairpin–oligo(U) sequence of intrinsic termination sites, the +identification of conserved patterns in ρ -dependent terminators has +proven more difficult. + + +How does ρ promote the termination of RNA synthesis? A key clue is the +finding that ρ is hexameric and hydrolyzes ATP in the presence of +single-stranded RNA but not in the presence of DNA or duplex RNA. +Thus ρ is a helicase, homologous to the hexameric helicases that we +encountered in our discussion of DNA replication (Section 28.1). The +role of ρ in the termination of transcription in bacteria is as follows +( **Figure 29.14** ): + + +The ρ protein is brought into action by sequences located in the +nascent RNA that are rich in cytosine and poor in guanine. + + +A stretch of nucleotides is bound in such a way that the RNA passes +through the center of the structure. + + +The helicase activity of ρ enables the protein to pull the nascent +RNA while pursuing RNA polymerase. + + +When ρ catches RNA polymerase at the transcription bubble, it +breaks the RNA–DNA hybrid by functioning as an RNA–DNA +helicase. + + + + + +Proteins in addition to ρ may promote termination. For example, the +NusA protein enables RNA polymerase in _E. coli_ to recognize a +characteristic class of termination sites. A common feature of + +transcription termination, whether it relies on a protein or not, is that + + +the functioning signals lie in newly synthesized RNA rather than in the + +_._ +DNA template + + +## **29.3 Transcription Is Highly** **Regulated** + +As we will see in Chapter 31, the level at which different genes are +transcribed is highly regulated. Regulated gene expression is critical for +the development of multicellular organisms, the differentiation of +various cell types, and the response of bacteria to changes in their +environment. Here, we will discuss a few examples of how transcription +can be controlled. + +#### **Alternative sigma subunits in bacteria** **control transcription in response to changes** **in conditions** + + +As noted above, the σ factor allows for the specific binding of the +bacterial RNA polymerase to a promoter site on the template DNA. _E._ +_coli_ has seven distinct σ factors for recognizing several types of +promoter sequences in _E. coli_ DNA. The type that recognizes the + + +consensus sequences described earlier is called because it has a + + +mass of 70 kDa. A different σ factor comes into play when the + + +temperature is raised abruptly. _E. coli_ responds by synthesizing, + + +which recognizes the promoters of so-called _heat-shock genes_ . These +promoters exhibit sequences that are somewhat different from the + +sequence for standard promoters ( **Figure 29.15** ). The increased +transcription of heat-shock genes leads to the coordinated synthesis of a +series of protective proteins. Other σ factors respond to environmental +conditions, such as nitrogen starvation. These findings demonstrate + + +that σ plays the key role in determining when and where RNA +polymerase initiates transcription. + + + +Some other bacteria contain a much larger number of σ factors. For +example, the genome of the soil bacterium _Streptomyces coelicolor_ +encodes more than 60 σ factors recognized on the basis of their amino +acid sequences. This repertoire allows these cells to adjust their geneexpression programs to the wide range of conditions, with regard to +nutrients and competing organisms, that they may experience. + +#### **Some messenger RNAs directly sense** **metabolite concentrations** + + +As we shall explore in Chapter 31, the expression of many genes is +controlled in response to the concentrations of metabolites and +signaling molecules within cells. One set of control mechanisms found +in both prokaryotes and eukaryotes depends on the remarkable ability +of some mRNA molecules to form secondary structures that are capable +of directly binding small molecules. These structures are termed +**riboswitches** . + + +Consider a riboswitch that controls the synthesis of genes that +participate in the biosynthesis of riboflavin in the bacterium _Bacillus_ +_subtilis_ ( **Figure 29.16** ). When flavin mononucleotide (FMN), a key +intermediate in riboflavin biosynthesis, is present at high +concentration, it binds to the RNA transcript. Binding of FMN to the +transcript induces a hairpin structure that favors premature +termination. By trapping the RNA transcript in this terminationfavoring conformation, FMN prevents the production of functional fulllength mRNA. However, when FMN is present at low concentration, it +does not readily bind to the mRNA. Without FMN bound, the transcript +adopts an alternative conformation without the terminator hairpin, +allowing the production of the full-length mRNA. The occurrence of +riboswitches serves as a vivid illustration of how RNAs are capable of +forming elaborate, functional structures, though in the absence of +specific information we tend to depict them as simple lines. + + + + +#### **Control of transcription in eukaryotes is** **highly complex** + +We turn now to transcription in eukaryotes, a much more complex +process than in bacteria. Eukaryotic cells have a remarkable ability to +regulate precisely the time at which each gene is transcribed and how +much RNA is produced. This ability led to the evolution of multicellular +eukaryotes with distinct tissues. That is, multicellular eukaryotes use +differential transcriptional regulation to create different cell types. + + +Gene expression is influenced by three important characteristics unique +to eukaryotes: the nuclear membrane, complex transcriptional +regulation, and RNA processing. + + +1. _The nuclear membrane allows transcription and translation to take place_ + +_in different cellular compartments._ Transcription takes place in the +membrane-bound nucleus, whereas translation takes place outside +the nucleus in the cytoplasm. In bacteria, the two processes are +closely coupled ( **Figure 29.17** ). Indeed, the translation of bacterial +mRNA begins while the transcript is still being synthesized. The +spatial and temporal separation of transcription and translation +enables eukaryotes to regulate gene expression in much more +intricate ways, contributing to the richness of eukaryotic form and +function. + + +2. _A variety of types of promoter elements enables complex transcriptional_ + +_regulation_ . Like bacteria, eukaryotes rely on conserved sequences +in DNA to regulate the initiation of transcription. But bacteria have +only three promoter elements (the, and UP elements), +whereas eukaryotes use a variety of types of promoter elements, +each identified by its own conserved sequence. Not all possible +types will be present together in the same promoter. In eukaryotes, +elements that regulate transcription can be found upstream or +downstream of the start site and sometimes at distances much +farther from the start site than in prokaryotes. For example, +enhancer elements located on DNA far from the start site increase +the promoter activity of specific genes. + + +3. _The degree of RNA processing is much greater in eukaryotes than in_ + +_bacteria_ . Although both bacteria and eukaryotes modify RNA, +eukaryotes very extensively process nascent RNA destined to +become mRNA. This processing includes modifications to both +ends and, most significantly, splicing out segments of the primary +transcript. RNA processing is described in Section 29.4. + +#### **Eukaryotic DNA is organized into chromatin** + + +Whereas bacterial genomic DNA is relatively accessible to the proteins +involved in transcription, eukaryotic DNA is packaged into **chromatin**, a +complex formed between the DNA and a particular set of proteins. +Chromatin compacts and organizes eukaryotic DNA, and its presence +has dramatic consequences for gene regulation. Although the principles +for the construction of chromatin are relatively simple, the chromatin +structure for a complete genome is quite complicated. Importantly, in +any given eukaryotic cell, some genes and their associated regulatory +regions are relatively accessible for transcription and regulation, +whereas other genes are tightly packaged, less accessible, and therefore +inactive. Eukaryotic gene regulation frequently requires the +manipulation of chromatin structure. + + +Chromatin viewed with the electron microscope has the appearance of +beads on a string ( **Figure 29.18** ). Partial digestion of chromatin with +DNase exposes these particles, which consist of fragments of DNA (the +“string”) wrapped around octamers of proteins called _histones_ (the +“beads”). The complex formed by a histone octamer and a 145-bp DNA +fragment is called the **nucleosome** ( **Figure 29.19** ). + + +**FIGURE 29.18 Eukaryotic chromatin structure resembles beads on a string.** In this +electron micrograph of chromatin, the “beads” correspond to DNA complexed with +specific proteins into nucleosomes. Each bead has a diameter of approximately 100 +Å. + + +The overall structure of the nucleosome was revealed through electron +microscopic and x-ray crystallographic studies pioneered by Aaron Klug +and his colleagues. More recently, the three-dimensional structures of +reconstituted nucleosomes have been determined to higher resolution +by x-ray diffraction methods. The histone octamer is a complex of four +different types of histones (H2A, H2B, H3, and H4) that are homologous +and similar in structure. + + +The eight histones in the core are arranged into a tetramer + + +and a pair of H2A–H2B dimers. The tetramer and dimers come together +to form a left-handed superhelical ramp around which the DNA wraps. +In addition, each histone has an amino-terminal tail that extends out +from the core structure. These tails are flexible and contain many lysine +and arginine residues. As we shall see in Chapter 31, covalent +modifications of these tails play an essential role in regulating gene +expression. + + +#### **Three types of RNA polymerase synthesize** **RNA in eukaryotic cells** + +In bacteria, RNA is synthesized by a single kind of polymerase. In +contrast, the nucleus of a typical eukaryotic cell contains three types of +RNA polymerase differing in template specificity and location in the +nucleus ( **Table 29.2** ). The three polymerases are named for the order in +which they were discovered, which has no bearing on the relative +importance of their function. We will discuss them in an order that +reflects their similarities in localization, function, and regulation. We +will emphasize RNA polymerase II, since it transcribes all of the +protein-coding genes and has therefore been the focus of much +research investigating transcriptional mechanisms. + + +**TABLE 29.2 Eukaryotic RNA polymerases** + + + + + + + +I Nucleolus 18S, 5.8S, and 28S rRNA Insensitive + + + +II Nucleoplas + +m + + +III Nucleoplas + +m + + + +mRNA precursors and snRNA Strongly inhibited + + +tRNA and 5S rRNA Inhibited by high concentrations + + + +_RNA polymerase I_ is located in specialized structures within the nucleus +called nucleoli, where it transcribes the tandem array of genes for 18S, +5.8S, and 28S rRNA. The other rRNA molecule (5S rRNA) and all the +tRNA molecules are synthesized by _RNA polymerase III_, which is located +in the nucleoplasm rather than in nucleoli. _RNA polymerase II_, which + + +also is located in the nucleoplasm, synthesizes the precursors of mRNA +as well as several small RNA molecules, such as those of the splicing +apparatus and many of the precursors to small regulatory RNAs. All +three of the polymerases are large proteins, containing from 8 to 14 +subunits and having total molecular masses greater than 500 kDa (or 0.5 +MDa), and it is likely that they evolved from a single enzyme that was +present in a common ancestor of eukaryotes, bacteria, and archaea. In +fact, many components of the eukaryotic transcriptional machinery +evolved from those in a common ancestor. + + +Although all eukaryotic RNA polymerases are homologous to one +another and to prokaryotic RNA polymerases, RNA polymerase II + +**-** +contains a unique **carboxyl** **terminal domain** (CTD) on the 220-kDa +subunit; this domain is unusual because it contains multiple repeats of +a YSPTSPS consensus sequence. The activity of RNA polymerase II is +regulated by phosphorylation, mainly on the serine residues of the CTD. + + +The different polymerases were originally distinguished through their +variable responses to the toxin α -amanitin, a cyclic octapeptide that +contains several modified amino acids and is produced by a genus of +poisonous mushroom ( **Figure 29.20** ). α -Amanitin binds very tightly + + +to RNA polymerase II and thereby blocks the elongation + + +phase of RNA synthesis. Higher concentrations of α -amanitin (1 μ M) +inhibit RNA polymerase III, whereas RNA polymerase I is insensitive to +this toxin. This pattern of sensitivity is highly conserved throughout the +animal and plant kingdoms. + + +**FIGURE 29.20** **α** **-Amanitin is produced by poisonous mushrooms in the genus** +_**Amanita**_ **.** Pictured is _Amanita phalloides_, also called the _death cap_ or the _destroying_ +_angel_ . + + +Finally, eukaryotic polymerases differ from each other in the promoters +to which they bind. Eukaryotic genes, like prokaryotic genes, require +promoters for transcription initiation. Like prokaryotic promoters, +eukaryotic promoters consist of conserved sequences that attract the +polymerase to the start site. However, eukaryotic promoters differ +distinctly in sequence and position, depending on the type of RNA +polymerase that binds to them ( **Figure 29.21** ). + + +_The promoter sequences for RNA polymerase I are located in stretches of_ +_DNA separating the ribosomal DNA (rDNA) it transcribes._ These rRNA +genes are arranged in several hundred tandem repeats, each +containing a copy of each of three rRNA genes. At the +transcriptional start site lies a TATA-like sequence called the +_ribosomal initiator element_ (rInr). Farther upstream, 150 to 200 bp +from the start site, is the _upstream promoter element_ (UPE). Both +elements aid transcription by binding proteins that recruit RNA +polymerase I. + + +_Promoters for RNA polymerase II, like prokaryotic promoters, include a_ +_set of consensus sequences that define the start site and recruit the_ +_polymerase._ However, the promoter can contain any combination of +a number of possible consensus sequences. Unique to eukaryotes, +they also include enhancer elements that can be more than 1 kb +from the start site. + + +_Promoters for RNA polymerase III are within the transcribed sequence,_ +_downstream of the start site_ . This is contrast to promoters for RNA +polymerase I and II, which are _upstream_ of the transcription start +site. There are two types of intergenic promoters for RNA +polymerase III. Type I promoters, found in the 5S rRNA gene, +contain two short, conserved sequences, the A block and the C +block. Type II promoters, found in tRNA genes, consist of two 11-bp +sequences, the A block and the B block, situated about 15 bp from +either end of the gene. + + +#### **Three common elements can be found in the** **RNA polymerase II promoter region** + +RNA polymerase II transcribes all of the protein-coding genes in +eukaryotic cells. Promoters for RNA polymerase II, like those for +bacterial polymerases, are generally located upstream of the start site +for transcription. Because these sequences are on the _same_ molecule of +DNA as the genes being transcribed, they are called _cis-acting elements._ + + +1. The most commonly recognized cis-acting element for genes + +transcribed by RNA polymerase II is called the **TATA box** on the +basis of its consensus sequence ( **Figure 29.22** ). The TATA box is +usually found between positions and . Note that the + +eukaryotic TATA box closely resembles the prokaryotic +sequence (TATAAT) but is farther from the start site. The mutation +of a single base in the TATA box markedly impairs promoter +activity. Thus, the precise sequence, not just a high content of AT +pairs, is essential. + + + + + +2. The TATA box is often paired with an _initiator element_ (Inr), a + +sequence found at the transcriptional start site, between positions + +and . This sequence defines the start site because the other + + +promoter elements are at variable distances from that site. Its +presence increases transcriptional activity. + + +3. A third element, the _downstream core promoter element_ (DPE), is + +commonly found in conjunction with the Inr in transcripts that +lack the TATA box. In contrast with the TATA box, the DPE is found +downstream of the start site, between positions and . + +#### **Regulatory cis-acting elements are** **recognized by different mechanisms** + + +Additional regulatory sequences are located between and . + + +Many promoters contain a _CAAT box_, and some contain a _GC box_ ( **Figure** +**29.23** ). Constitutive genes (genes that are continuously expressed rather +than regulated) tend to have GC boxes in their promoters. The positions +of these upstream sequences vary from one promoter to another, in +contrast with the quite constant location of the region in +prokaryotes. Another difference is that the CAAT box and the GC box +can be effective when present on the template (antisense) strand, unlike +the region, which must be present on the coding (sense) strand. + + +These differences between prokaryotes and eukaryotes correspond to +fundamentally different mechanisms for the recognition of cis-acting +elements. The and sequences in prokaryotic promoters are +binding sites for RNA polymerase and its associated σ factor. In +contrast, the TATA, CAAT, and GC boxes and other cis-acting elements in +eukaryotic promoters are recognized by proteins other than RNA +polymerase itself. + + +#### **The TFIID protein complex initiates the** **assembly of the active transcription complex** **in eukaryotes** + +Cis-acting elements constitute only part of the puzzle of eukaryotic gene +expression. **Transcription factors** that bind to these elements also are +required. For example, RNA polymerase II is guided to the start site by a +set of transcription factors known collectively as _TFII_ ( _TF_ stands for +transcription factor, and _II_ refers to RNA polymerase II). Individual TFII +factors are called TFIIA, TFIIB, and so on. + + +In TATA-box promoters, the key initial event is the recognition of the +TATA box by the TATA-box-binding protein (TBP), a 30-kDa component +of the 700-kDa TFIID complex. In TATA-less promoters, other proteins in +the TFIID complex bind the core promoter elements; however, because +less is known about these interactions, we will consider only the TATAbox–TBP binding interaction. TBP binds times as tightly to the TATA + + +box as to nonconsensus sequences; the dissociation constant of the +TBP–TATA-box complex is approximately 1 nM. + + +The TATA box of DNA binds to the concave surface of TBP, inducing +large conformational changes in the bound DNA ( **Figure 29.24** ). The +double helix is substantially unwound to widen its minor groove, +enabling it to make extensive contact with the antiparallel β strands on +the concave side of TBP. Hydrophobic interactions are prominent at this +interface. Four phenylalanine residues, for example, are intercalated +between base pairs of the TATA box. The flexibility of AT-rich sequences +is generally exploited here in bending the DNA. Immediately outside the +TATA box, classical B-DNA resumes. The TBP–TATA-box complex is +distinctly asymmetric, a property that is crucial for specifying a unique +start site and ensuring that transcription proceeds unidirectionally. + + +TBP bound to the TATA box is the heart of the initiation complex ( **Figure** +**29.25** ). The surface of the TBP saddle provides docking sites for the +binding of other components, with additional transcription factors +assembling on this nucleus in a defined sequence. TFIIA is recruited, +followed by TFIIB; then TFIIF, RNA polymerase II, TFIIE, and TFIIH join +the other factors to form a complex called the _pre-initiation complex_ +(PIC). + + +These additional transcription factors play specific roles in this +complex. As we saw above, TFIID recognizes core promoter elements +and is central to the assembly process. While TFIIA is not essential for +the assembly or function of the PIC in vitro, it may aid in the binding of +TFIID to the DNA. TFIIB is a DNA-binding protein that recognizes +specific cis-acting promoter elements called _B recognition elements_, +which are often found near the TATA box. TFIIF aids in the recruitment +of polymerase II, while TFIIE brings TFIIH to the complex. TFIIH is a +multisubunit complex with helicase and protein kinase activities, both +of which are critical in the initiation of transcription. The helicase +activity unwinds the DNA template, and the kinase activity +phosphorylates specific amino acids in the CTD of polymerase II. + + +During the formation of the PIC, the carboxyl-terminal domain (CTD) is +unphosphorylated and plays a role in transcription regulation through +its binding to an enhancer-associated complex called _mediator_ (Section +31.4). Phosphorylation of the CTD by TFIIH marks the transition from +initiation to elongation. The phosphorylated CTD stabilizes +transcription elongation by RNA polymerase II and recruits RNAprocessing enzymes that act during the course of elongation. The +importance of the carboxyl-terminal domain is highlighted by the +finding that yeast cells containing mutant polymerase II with fewer than +10 repeats in the CTD are not viable. + + +The PIC described above initiates transcription at a low (basal) +frequency, and the transcription factors associated with it are referred + + +to as _basal_ or +_general_ transcription factors. Additional transcription +factors that bind to other sites are required to achieve a high rate of +mRNA synthesis. Their role is to selectively stimulate _specific g_ enes. In +summary, transcription factors and other proteins that bind to +regulatory sites on DNA can be regarded as passwords that +cooperatively open multiple locks, giving RNA polymerase access to + +_._ +specific genes + +###### **Self–Check Question** + + +The function of the σ subunit of _E. coli_ RNA polymerase is analogous to the function of +general transcription factors in eukaryotes. Briefly describe their common function. + +#### **Enhancer sequences can stimulate** **transcription at start sites thousands of** **bases away** + + +The activities of many promoters in higher eukaryotes are greatly +**enhancer** . +increased by another type of cis-acting element called an + + +Enhancer sequences have no promoter activity of their own yet can +exert their stimulatory actions over distances of several thousand +base pairs. + + +Enhancers can be upstream, downstream, or even in the middle of +a transcribed gene _._ + + +Enhancers are effective when present on either the coding or +noncoding DNA strand _._ + + +A particular enhancer is effective only in certain cells; for example, +the immunoglobulin enhancer functions in B lymphocytes but not +elsewhere. + + +Cancer can result if the relation between genes and enhancers is + + +disrupted. In Burkitt lymphoma and B-cell leukemia, a chromosomal +translocation brings the proto-oncogene _myc_ (a transcription factor +itself) under the control of a powerful immunoglobulin enhancer. The +consequent dysregulation of the _myc_ gene is hypothesized to play a role +in the progression of the cancer. + + +The discovery of promoters and enhancers has allowed us to gain a +better understanding of how genes are selectively expressed in +eukaryotic cells. The regulation of eukaryotic gene transcription, +discussed in Chapter 31, is the fundamental means of controlling gene +expression. + + +## **29.4 Some RNA Transcription** **Products Are Processed** + +Virtually all the initial products of eukaryotic transcription are further +processed, and even some prokaryotic transcripts are modified. As we +will see next, the particular processing steps and the factors taking part +vary according to the type of RNA precursor and the type of RNA +polymerase that produced it. + +#### **Precursors of transfer and ribosomal RNA** **are cleaved and chemically modified after** **transcription** + + +In bacteria, messenger RNA molecules undergo little or no modification +after synthesis by RNA polymerase. Indeed, many mRNA molecules are +translated while they are being transcribed. In contrast, transfer RNA +(tRNA) and ribosomal RNA (rRNA) molecules are generated by + +_._ +modifications of nascent RNA chains + + +For +_The transcript can be cleaved at specific sites along its sequence._ +example, in _E. coli_, the three rRNAs and a tRNA are excised from a +single primary RNA transcript that also contains spacer regions +( **Figure 29.26** ). Other transcripts contain arrays of several kinds of +tRNA or several copies of the same tRNA. The nucleases that cleave +and trim these precursors of rRNA and tRNA are highly precise. + + +_Ribonuclease P_ (RNase P), for example, generates the correct + + +terminus of all tRNA molecules in _E. coli._ Sidney Altman and his +coworkers showed that this interesting enzyme contains a +catalytically active RNA molecule. _Ribonuclease III_ (Rnase III) +excises 5S, 16S, and 23S rRNA precursors from the primary + + +transcript by cleaving double-helical hairpin regions at specific +sites. + + + + + +_Nucleotides can be added to the termini of some RNA chains_ . For +example, CCA, a terminal sequence required for the function of all + + +tRNAs, is added to the ends of tRNA molecules for which this + + +terminal sequence is not encoded in the DNA. The enzyme that +catalyzes the addition of CCA is atypical for an RNA polymerase in +that it does not use a DNA template. + + +_Bases and ribose units of RNAs can be modified._ For example, some +bases of rRNA are methylated. Furthermore, all tRNA molecules +contain unusual bases formed by the enzymatic modification of a +standard ribonucleotide in a tRNA precursor. For example, +uridylate residues are modified after transcription to form +ribothymidylate and pseudouridylate. These modifications generate +diversity, allowing greater structural and functional versatility. + + +#### **RNA polymerase I produces three ribosomal** **RNAs** + +Several RNA molecules are key components of ribosomes. In +eukaryotes, RNA polymerase I transcription produces a single precursor +(45S in mammals) that encodes three RNA components of the ribosome: +the 18S rRNA, the 28S rRNA, and the 5.8S rRNA ( **Figure 29.27** ). + + + + + +The 18S rRNA is the RNA component of the small ribosomal subunit +(40S), and the 28S and 5.8S rRNAs are two RNA components of the large +ribosomal subunit (60S). The other RNA component of the large + + +ribosomal subunit, the 5S rRNA, is transcribed by RNA polymerase III as +a separate transcript. Processing of the precursor proceeds as follows: + + +First, the nucleotides of the pre-rRNA sequences destined for the +ribosome undergo extensive modification, on both ribose and base +components, directed by many **small nucleolar** +**ribonucleoproteins (snoRNPs)**, each of which consists of one +snoRNA and several proteins. + + +The pre-rRNA is then assembled with ribosomal proteins, as guided +by processing factors, to form a large ribonucleoprotein. For +instance, the small-subunit (SSU) processome is required for 18S +rRNA synthesis and can be visualized in electron micrographs as a + + +terminal knob at the ends of the nascent rRNAs ( **Figure 29.28** ). + + +Finally, rRNA cleavage (sometimes coupled with additional +processing steps) releases the mature rRNAs assembled with +ribosomal proteins as ribosomes. Like those of RNA polymerase I +transcription itself, most of these processing steps take place in the +cell’s nucleolus. + +#### **RNA polymerase III produces transfer RNAs** + + +Eukaryotic tRNA transcripts are among the most processed of all RNA + + +polymerase III transcripts. Like those of prokaryotic tRNAs, the + + +leader is cleaved by RNase P, the trailer is removed, and CCA is added + + +by the CCA-adding enzyme ( **Figure 29.29** ). Eukaryotic tRNAs are also +heavily modified on base and ribose moieties; these modifications are +important for function. In contrast with prokaryotic tRNAs, many +eukaryotic pre-tRNAs are also spliced by an endonuclease and a ligase +to remove an intron. + + +#### **The product of RNA polymerase II, the pre-** **mRNA transcript, acquires a cap and a** + + +#### **poly(A) tail** + +Perhaps the most extensively studied transcription product is the +product of RNA polymerase II: most of this RNA will be processed to +mRNA. The immediate product of RNA polymerase II is sometimes + +**-** +referred to as precursor-to-messenger RNA, or **pre** **mRNA** . Most premRNA molecules are spliced to remove the introns, which we will + + +discuss in greater detail below. In addition, both the and the ends + +are modified, and both modifications are retained as the pre-mRNA is +converted into mRNA. + + +As in prokaryotes, eukaryotic transcription usually begins with A or G. + + +However, the triphosphate end of the nascent RNA chain is + +immediately modified: + + +First, a phosphoryl group is released by hydrolysis. + + +The diphosphate end then attacks the α -phosphorus atom of GTP + + +to form a very unusual triphosphate linkage. This distinctive + + +terminus is called a **cap** ( **Figure 29.30** ). + + + The N-7 nitrogen of the terminal guanine is then methylated by _S_ +adenosylmethionine to form cap 0. The adjacent riboses may be +methylated to form cap 1 or cap 2. + + +Caps contribute to the stability of mRNAs by protecting their ends + + +from phosphatases and nucleases. In addition, caps enhance the +translation of mRNA by eukaryotic protein-synthesizing systems. +Transfer RNA and ribosomal RNA molecules, in contrast with +messenger RNAs and with small RNAs that participate in splicing, do +not have caps. + + +As mentioned earlier, pre-mRNA is also modified at the end. Most + + +eukaryotic mRNAs contain a string of adenine nucleotides — a **poly(A)** +**tail** — at that end. This poly(A) tail is added _after_ transcription has +ended, since the DNA template does not encode this sequence. Indeed, +the nucleotide preceding poly(A) is not the last nucleotide to be +transcribed. Some primary transcripts contain hundreds of nucleotides + + +beyond the end of the mature mRNA. + + +How is the end of the pre-mRNA given its final form? Eukaryotic + +primary transcripts are cleaved by a specific endonuclease that +recognizes the sequence AAUAAA ( **Figure 29.31** ). Cleavage does not take + + +place if this sequence or a segment of some 20 nucleotides on its side + + +is deleted. The presence of internal AAUAAA sequences in some mature +mRNAs indicates that AAUAAA is only part of the cleavage signal; its +context also is important. After cleavage of the pre-RNA by the +endonuclease, a _poly(A) polymerase_ adds about 250 adenylate residues to + + +the end of the transcript; ATP is the donor in this reaction. + + + + + +The role of the poly(A) tail is still not firmly established despite much +effort. However, evidence is accumulating that it enhances translation +efficiency and the stability of mRNA. Blocking the synthesis of the +poly(A) tail by exposure to -deoxyadenosine (cordycepin) does not + + +interfere with the synthesis of the primary transcript. Messenger RNA + + +without a poly(A) tail can be transported out of the nucleus. However, an +mRNA molecule without a poly(A) tail is usually much less effective as a +template for protein synthesis than one with a poly(A) tail. Indeed, +some mRNAs are stored in an unadenylated form and receive the +poly(A) tail only when translation is imminent. The half-life of an mRNA +molecule may be determined in part by the rate of degradation of its +poly(A) tail. + +#### **Sequences at the ends of introns specify** **splice sites in mRNA precursors** + + +Most genes in higher eukaryotes are composed of exons and introns +(Section 8.7). The introns must be excised and the exons linked to form +the final mRNA in a process called **RNA splicing** . This splicing must be +exquisitely sensitive; splicing just one nucleotide upstream or +downstream of the intended site would create a one-nucleotide shift, + + +which would alter the reading frame on the side of the splice to give + + +an entirely different amino acid sequence, likely including a premature +stop codon. Thus, the correct splice site must be clearly marked. + + +Does a particular sequence denote the splice site? The sequences of +thousands of intron–exon junctions within RNA transcripts are known. +In eukaryotes from yeast to mammals, these sequences have a common +structural motif: the intron begins with GU and ends with AG. The + + +consensus sequence at the splice in vertebrates is AGGUAAGU, where + + +the GU is invariant ( **Figure 29.32** ). At the end of an intron, the + + +consensus sequence is a stretch of 10 pyrimidines (U or C; termed the +_polypyrimidine tract_ ), followed by any base, then by C, and ending with +the invariant AG. Introns also have an important internal site located + + +between 20 and 50 nucleotides upstream of the splice site; it is called + + +the _branch site_ for reasons that will be evident shortly. In yeast, the +branch-site sequence is nearly always UACUAAC, whereas in mammals +a variety of sequences are found. + + +The and splice sites and the branch site are essential for + + +determining where splicing takes place. Mutations in each of these +three critical regions lead to aberrant splicing. Introns vary in length +from 50 to 10,000 nucleotides, and so the splicing machinery may have + +to find the site several thousand nucleotides away. Specific sequences + + +near the splice sites (in both the introns and the exons) play an +important role in splicing regulation, particularly in designating splice +sites when there are many alternatives. Researchers are currently +attempting to determine the factors that contribute to splice-site +selection for individual mRNAs. Despite our knowledge of splice-site +sequences, predicting pre-mRNAs and their protein products from +genomic DNA sequence information remains a challenge. + + +#### **Splicing consists of two sequential** **transesterification reactions** + +The splicing of nascent mRNA molecules is a complicated process. It +requires the cooperation of several small RNAs and proteins that form a +large complex called a **spliceosome** . However, the chemistry of the +splicing process is simple. Splicing begins with the cleavage of the + + +phosphodiester bond between the upstream exon (exon 1) and the + + +end of the intron ( **Figure 29.33** ). The attacking group in this reaction is + + +the group of an adenylate residue in the branch site. A + + +phosphodiester bond is formed between this A residue and the + +terminal phosphate of the intron in a transesterification reaction. + + +Note that this adenylate residue is also joined to two other nucleotides + + +by normal phosphodiester bonds ( **Figure 29.34** ). Hence, a branch + + +is generated at this site, and a lariat (loop) intermediate is formed. + + +The terminus of exon 1 then attacks the phosphodiester bond + + +between the intron and exon 2. In another transesterification reaction, +exons 1 and 2 become joined, and the intron is released in lariat form. +Splicing is thus accomplished by two transesterification reactions rather +than by hydrolysis followed by ligation. + + +Both transesterification reactions are promoted by the pair of bound +magnesium ions, in reactions reminiscent of those for DNA and RNA + +polymerases. The first reaction generates a free group at the + + + end of exon 1, and the second reaction links this group to the + + +phosphate of exon 2. The number of phosphodiester bonds stays the +same during these steps, which is crucial because it allows the splicing +reaction itself to proceed without an energy source such as ATP or GTP. + +#### **Small nuclear RNAs in spliceosomes catalyze** **the splicing of mRNA precursors** + + +The nucleus contains many types of small RNA molecules with fewer +than 300 nucleotides, referred to as **small nuclear RNAs (snRNAs)** . A +few of them — designated U1, U2, U4, U5, and U6 — are essential for +splicing mRNA precursors. The secondary structures of these RNAs are +highly conserved in organisms ranging from yeast to human beings. + + +snRNA molecules are associated with specific proteins to form +complexes termed **small nuclear ribonucleoproteins (snRNPs)** ; +investigators often speak of them as “snurps” ( **Table 29.3** ). SnRNPs and +their role in RNA splicing were discovered by Joan Steitz and Michael +Lerner in 1980. One major piece of evidence suggesting a role for +snRNPs in splicing was the base complementarity between portions of +the U1 snRNA and the splice sites found in the unprocessed mRNAs. + + +**TABLE 29.3 Roles of small nuclear ribonucleoproteins (snRNPs) in the** +**splicing of mRNA precursors** + + + + + + + + + +U1 165 Binds the splice site + + +U2 185 Binds the branch site + + +U5 116 +Binds the splice site and then the splice site + + +U4 145 Masks the catalytic activity of U6 + + +U6 106 Catalyzes splicing + + +SnRNPs associate with hundreds of other proteins (called _splicing_ +_factors_ ) and the mRNA precursors to form the large (60S) spliceosomes. +The large and dynamic nature of the spliceosome made the +determination of the detailed three-dimensional structure a great +challenge. However, with the maturation of cryo-electron microscopy +(Section 4.5), the structures of spliceosomes from several species in a +number of different stages of their function have been determined +( **Figure 29.35** ). These structures have added to our understanding of the +splicing process ( **Figure 29.36** ). + + +1. Splicing begins with the recognition of the splice site by the U1 + + +snRNP. U1 snRNA contains a highly conserved six-nucleotide +sequence, not covered by protein in the snRNP, that base-pairs to + + +the splice site of the pre-mRNA. This binding initiates + + +spliceosome assembly on the pre-mRNA molecule. + + +2. U2 snRNP then binds the branch site in the intron by base-pairing + +between a highly conserved sequence in U2 snRNA and the premRNA. U2 snRNP binding requires ATP hydrolysis. + + +3. A preassembled U4-U5-U6 tri-snRNP joins this complex of U1, U2, + +and the mRNA precursor to form the spliceosome. This association +also requires ATP hydrolysis. Experiments with a reagent that crosslinks neighboring pyrimidines in base-paired regions revealed that + + +in this assembly U5 interacts with exon sequences in the splice + + +exon. +site and subsequently with the + + +4. Next, U6 disengages from U4 and undergoes an intramolecular + +rearrangement that permits base-pairing with U2 as well as + + +interaction with the end of the intron, displacing U1 and U4 from + + +the spliceosome. U4 serves as an inhibitor that masks U6 until the +specific splice sites are aligned. The catalytic center includes two +bound magnesium ions bound primarily by phosphate groups from +the U6 RNA ( **Figure 29.37** ). + + +5. These rearrangements result in the first transesterification + + +reaction, cleaving the exon and generating the lariat + + +intermediate. + + +6. Further rearrangements of RNA in the spliceosome facilitate the + +second transesterification. In these rearrangements, U5 aligns the + + +free exon with the exon such that the -hydroxyl group of the + + +exon is positioned to make a nucleophilic attack on the splice + + +site to generate the spliced product. U2, U5, and U6 bound to the +excised lariat intron are released, completing the splicing reaction. + + +Many of the steps in the splicing process require ATP hydrolysis. How is +the free energy associated with ATP hydrolysis used to power splicing? +To achieve the well-ordered rearrangements necessary for splicing, +ATP-powered RNA helicases must unwind RNA helices and allow +alternative base-pairing arrangements to form. Thus, two features of the +splicing process are noteworthy. First, RNA molecules play key roles in +directing the alignment of splice sites and in carrying out catalysis. +Second, ATP-powered helicases unwind RNA duplex intermediates that +facilitate catalysis and induce the release of snRNPs from the mRNA. + +#### **Mutations that affect pre-mRNA splicing** **cause disease** + + +Mutations in either the pre-mRNA (cis-acting) or the splicing factors +(trans-acting) can cause defective pre-mRNA splicing that manifests in +disease. In fact, mutations affecting splicing have been estimated to +cause at least 15% of all genetic diseases. We will look at two examples +here. + + +First, we will consider the possible effects of cis-acting mutations on +hemoglobin function. Mutations in the pre-mRNA cause some forms of +thalassemia, a group of hereditary anemias characterized by the +defective synthesis of hemoglobin (Section 3.3). Cis-acting mutations + + +or +that cause aberrant splicing can occur at the splice sites in + + +either of the two introns of the hemoglobin β chain or in its exons. + + +Typically, mutations in the splice site alter that site such that the + +splicing machinery cannot recognize it, forcing the machinery to find + + +another splice site in the intron and introducing the potential for a + + +premature stop codon. The defective mRNA is normally degraded rather +than translated. Alternatively, mutations in the intron itself may create a + + +new +splice site; in this case, either one of the two splice sites may be + + +recognized ( **Figure 29.38** ). Consequently, some normal protein can be +made, and so the disease is less severe. + + + + +Second, we will consider the possible effects of trans-acting mutations +on eyesight. Disease-causing mutations may also appear in splicing +factors. Retinitis pigmentosa is a disease of acquired blindness, first +described in 1857, with an incidence of 1/3500. About 5% of the +autosomal dominant form of retinitis pigmentosa is likely due to +mutations in the hPrp8 protein, a pre-mRNA splicing factor that is a +component of the U4-U5-U6 tri-snRNP. How a mutation in a splicing +factor that is present in all cells causes disease only in the retina is not +clear; nevertheless, retinitis pigmentosa is a good example of how +mutations that disrupt spliceosome function can cause disease. + +#### **Most human pre-mRNAs can be spliced in** **alternative ways to yield different proteins** + + +As a result of **alternative splicing**, different combinations of exons from +the same gene may be spliced into a mature RNA, producing distinct +forms of a protein for specific tissues, developmental stages, or +signaling pathways. What controls which splicing sites are selected? The +selection is determined by the binding of trans-acting splicing factors to +cis-acting sequences in the pre-mRNA. Most alternative splicing leads to +changes in the coding sequence, resulting in proteins with different +functions. + + +Alternative splicing provides a powerful mechanism for generating +protein diversity. It expands the versatility of genomic sequences +through combinatorial control. Consider a gene with five positions at +which splicing can take place. With the assumption that these + + +alternative splicing pathways can be regulated independently, a total of + +different mRNAs can be generated. + + +Sequencing of the human genome has revealed that most pre-mRNAs +are alternatively spliced, leading to a much greater number of proteins +than would be predicted from the number of genes. An example of +alternative splicing leading to the expression of two different proteins, +each in a different tissue, is provided by the gene encoding both +calcitonin and calcitonin-gene-related peptide (CGRP; **Figure 29.39** ). In +the thyroid gland, the inclusion of exon 4 in one splicing pathway +produces calcitonin, a peptide hormone that regulates calcium and +phosphorus metabolism. In neuronal cells, the exclusion of exon 4 in +another splicing pathway produces CGRP, a peptide hormone that acts +as a vasodilator. A single pre-mRNA thus yields two very different +peptide hormones, depending on cell type. + + + + + +In the above example, only two proteins result from alternative splicing; +however, in other cases, many more can be produced. An extreme + + +example is the _Drosophila_ pre-mRNA that encodes DSCAM, a neuronal +protein affecting axon connectivity. Alternative splicing of this premRNA has the potential to produce 38,016 different combinations of +exons, a greater number than the total number of genes in the +_Drosophila_ genome. However, only a fraction of these potential mRNAs +appear to be produced, owing to regulatory mechanisms that are not yet +well understood. + + +Several human diseases that can be attributed to defects in alternative +splicing are listed in **Table 29.4** . Further understanding of alternative +splicing and the mechanisms of splice-site selection will be crucial to +understanding how the proteome represented by the human genome is +expressed. + + +**TABLE 29.4 Selected human disorders attributed to defects in** +**alternative splicing** + + +Acute intermittent porphyria Porphobilinogen deaminase + + +Breast and ovarian cancer _BRCA1_ + + +Cystic fibrosis _CFTR_ + + +Frontotemporal dementia protein + + +Hemophilia A Factor VIII + + + +HGPRT deficiency (Lesch–Nyhan +syndrome) + + + +Hypoxanthine-guanine +phosphoribosyltransferase + + + +Leigh encephalomyelopathy Pyruvate dehydrogenase E1 α + + +Severe combined immunodeficiency Adenosine deaminase + + +Spinal muscle atrophy _SMN1_ or _SMN2_ + +#### **Transcription and mRNA processing are** **coupled** + + +Although we have described the transcription and processing of mRNAs +as separate events in gene expression, experimental evidence suggests +that the two steps are coordinated by the carboxyl-terminal domain of +RNA polymerase II. We have seen that the CTD consists of a unique +repeated seven-amino-acid sequence, YSPTSPS. Either,, or both +may be phosphorylated in the various repeats. The phosphorylation +state of the CTD is controlled by a number of kinases and phosphatases +and leads the CTD to bind many of the proteins having roles in RNA +transcription and processing. The CTD contributes to efficient +transcription by recruiting certain proteins to the pre-mRNA ( **Figure** +**29.40** ). These proteins include: + + +1. Capping enzymes, which methylate the guanine on the pre +mRNA immediately after transcription begins + + +2. Components of the splicing machinery, which initiate the excision + +of each intron as it is synthesized + + +3. An endonuclease that cleaves the transcript at the poly(A) addition + + +site, creating a free group that is the target for + + +adenylation + + +These events take place sequentially, directed by the phosphorylation +state of the CTD. + +#### **Small regulatory RNAs are cleaved from** **larger precursors** + + +Cleavage plays a role in the processing of small single-stranded RNAs +**microRNAs** . +(approximately 20–23 nucleotides) called MicroRNAs play + +. +key roles in gene regulation in eukaryotes, as we shall see in Chapter 31 +They are generated from initial transcripts produced by RNA +polymerase II and, in some cases, RNA polymerase III. These +transcripts fold into hairpin structures that are cleaved by specific +nucleases at various stages ( **Figure 29.41** ). The final single-stranded + + +RNAs are bound by regulatory proteins, where the RNAs help target the +regulation of specific genes. + + + + +#### **RNA editing can lead to specific changes in** **mRNA** + +Remarkably, the amino acid sequence information encoded by some +mRNAs is altered after transcription. This phenomenon is referred to as +**RNA editing**, a posttranscriptional change in the nucleotide sequence of +RNA that is caused by processes other than RNA splicing. RNA editing is +prominent in some systems; next, we will consider three examples. + + +RNA editing is key to the process of lipid transport by apolipoprotein B +(apo B). Apo B plays an important role in the transport of +triacylglycerols and cholesterol by forming an amphipathic spherical +shell around the lipids carried in lipoprotein particles (Section 27.3). +Apo B exists in two forms, a 512-kDa _apo B-100_ and a 240-kDa _apo B-48._ +The larger form, synthesized by the liver, participates in the transport of +lipids synthesized in the cell. The smaller form, synthesized by the + + +small intestine, carries dietary fat in the form of chylomicrons. Apo B-48 +contains the 2152 N-terminal residues of the 4536-residue apo B-100. +This truncated molecule can form lipoprotein particles but cannot bind +to the low-density-lipoprotein receptor on cell surfaces. + + +What is the relationship between these two forms of apo B? +Experiments revealed that a totally unexpected mechanism for +generating diversity is at work: the changing of the nucleotide sequence +of mRNA _after_ its synthesis ( **Figure 29.42** ) _._ A specific cytidine residue of +mRNA is deaminated to uridine, which changes the codon at residue +2153 from CAA (Gln) to UAA (stop). The deaminase that catalyzes this +reaction is present in the small intestine, but not in the liver, and is +expressed only at certain developmental stages. + + +RNA editing also plays a role in the regulation of postsynaptic receptors. +Glutamate opens cation-specific channels in the vertebrate central +nervous system by binding to receptors in postsynaptic membranes. +RNA editing changes a single glutamine codon (CAG) in the mRNA for +the glutamate receptor to the codon for arginine (CGG). The substitution + +of Arg for Gln in the receptor prevents, but not, from flowing + + +through the channel. + + +In trypanosomes (parasitic protozoans), a different kind of RNA editing +markedly changes several mitochondrial mRNAs. Nearly half the +uridine residues in these mRNAs are inserted by RNA editing. A guide +RNA molecule identifies the sequences to be modified, and a poly(U) tail +on the guide RNA donates uridine residues to the mRNAs undergoing +editing. + + +DNA sequences evidently do not always faithfully represent the +sequence of encoded proteins; crucial functional changes to mRNA can +take place. RNA editing is likely much more common than was formerly +thought. The chemical reactivity of nucleotide bases — including the +susceptibility to deamination that necessitates complex DNA-repair +mechanisms — has been harnessed as an engine for generating +molecular diversity at the RNA and, hence, protein levels. + + +## **29.5 The Discovery of Catalytic RNA** **Revealed a Unique Splicing** **Mechanism** + +RNAs form a surprisingly versatile class of molecules. As we have seen, +splicing is catalyzed largely by RNA molecules, with proteins playing a +secondary role. RNA is also a key component of ribonuclease P, which +catalyzes the maturation of tRNA by endonucleolytic cleavage of + + +nucleotides from the end of the precursor molecule. Finally, as we + + +shall see in Chapter 30, the RNA component of ribosomes is the catalyst +that carries out protein synthesis. + +#### **Some RNAs can promote their own splicing** + + +The versatility of RNA first became clear from observations of the +processing of ribosomal RNA in a single-cell eukaryote, a ciliated +protozoan in the genus _Tetrahymena._ In _Tetrahymena_, a 414-nucleotide +intron is removed from a 6.4-kb precursor to yield the mature 26S rRNA +molecule. + + +In an elegant series of studies of this splicing reaction, Thomas Cech +and his coworkers established that, in the absence of protein, the RNA +spliced itself to precisely excise the intron. Indeed, the RNA alone is +catalytic and, under certain conditions, is thus a **ribozyme** . More than +1500 similar introns have since been found in species as widely +dispersed as bacteria and eukaryotes, though not in vertebrates. + +**-** +Collectively, they are referred to as group I **self** **splicing introns** . + + +**THOMAS CECH** Fascinated by the structure of chromosomes, Thomas Cech pursued +this topic as a graduate student and postdoctoral fellow. He then accompanied his +wife, Dr. Carol Cech, to the University of Colorado in Boulder (UCB), where they had +both been offered faculty positions. They had met in chemistry class while they were +undergraduate students at Grinnell College. At UCB, Dr. Cech set out to purify +enzymes involved in the splicing of a particular RNA molecule, but he and his + + +coworkers soon discovered that the RNA molecule spliced itself. This fundamental +discovery of RNA catalysis, which changed our view of both modern biochemistry +and our evolutionary past, was recognized with the Nobel Prize in Chemistry in 1989. +Dr. Cech has remained at UCB, teaching with infectious enthusiasm throughout his +career. He also served as an investigator (and president from 2000 to 2009) of the +Howard Hughes Medical Institute (HHMI), a leading private funder of biomedical +research. + + +The self-splicing reaction in the group I intron requires an added +guanosine nucleotide ( **Figure 29.43** ). Nucleotides were originally +included in the reaction mixture because it was thought that ATP or GTP +might be needed as an energy source. Instead, the nucleotides were +found to be necessary as cofactors. The required cofactor proved to be a +guanosine unit, in the form of guanosine, GMP, GDP, or GTP. G +(denoting any one of these species) serves not as an energy source but +as an attacking group that becomes transiently incorporated into the + + +RNA. G binds to the RNA and then attacks the splice site to form a + + +phosphodiester bond with the end of the intron. This + + +transesterification reaction generates a group at the end of the + + +upstream exon. This group then attacks the splice site in a + +second transesterification reaction that joins the two exons and leads to +the release of the 414-nucleotide intron. + + +**FIGURE 29.43 Some introns are capable of self-splicing.** A ribosomal RNA precursor +representative of the group I introns, from the protozoan _Tetrahymena_, splices itself in the +presence of a guanosine cofactor (G, shown in green). A 414-nucleotide intron (red) is released in +the first splicing reaction. This intron then splices itself twice again to produce a linear RNA that +has lost a total of 19 nucleotides. This L19 RNA is catalytically active. + + +[Information from T. Cech, RNA as an enzyme. Copyright © 1986 by Scientific American, Inc. All rights reserved.] + + +Self-splicing depends on the structural integrity of the RNA precursor. +Much of the group I intron is needed for self-splicing. This molecule, +like many RNAs, has a folded structure formed by many double-helical +stems and loops ( **Figure 29.44** ), with a well-defined pocket for binding +the guanosine. Examination of the three-dimensional structure of a +catalytically active group I intron determined by x-ray crystallography +reveals the coordination of magnesium ions in the active site analogous +to that observed in protein enzymes such as DNA polymerase. + + +Analysis of the base sequence of the rRNA precursor suggested that the +splice sites are aligned with the catalytic residues by base-pairing + + +between the _internal guide sequence_ (IGS) in the intron and the and + +exons ( **Figure 29.45** ). The IGS first brings together the guanosine + + +cofactor and the splice site so that the group of G can make a + + +nucleophilic attack on the phosphorus atom at this splice site. The IGS +then holds the downstream exon in position for attack by the newly + + +formed group of the upstream exon. A phosphodiester bond is + + +formed between the two exons, and the intron is released as a linear +molecule. Like catalysis by protein enzymes, self-catalysis of bond +formation and breakage in this rRNA precursor is highly specific. + + +The finding of enzymatic activity in the self-splicing intron and in the +RNA component of RNase P has opened new areas of inquiry and +changed the way in which we think about molecular evolution. As +mentioned in an earlier chapter, the discovery that RNA can be a +catalyst as well as an information carrier suggests that an RNA world +may have existed early in the evolution of life, before the appearance of +DNA and protein. + + +Messenger RNA precursors in the mitochondria of yeast and fungi also +undergo self-splicing, as do some RNA precursors in the chloroplasts of +unicellular organisms such as _Chlamydomonas_ . Self-splicing reactions +can be classified according to the nature of the unit that attacks the +upstream splice site. Group I self-splicing is mediated by a guanosine +cofactor, as in _Tetrahymena_ . The attacking moiety in group II splicing is + +the group of a specific adenylate of the intron ( **Figure 29.46** ). + + +Group I and group II self-splicing resembles spliceosome-catalyzed +splicing in two respects. First, in the initial step, a ribose hydroxyl group + + +attacks the splice site. The newly formed terminus of the + + +upstream exon then attacks the splice site to form a phosphodiester + + +bond with the downstream exon. Second, both reactions are + + +transesterifications in which the phosphate moieties at each splice site +are retained in the products. The number of phosphodiester bonds stays + +constant. + + +Group II splicing is like the spliceosome-catalyzed splicing of mRNA + + +precursors in several additional ways. First, the attack at the splice + + +site is carried out by a part of the intron itself (the group of + + +adenosine) rather than by an external cofactor (G). Second, the intron is +released in the form of a lariat. Third, in some instances, the group II +intron is transcribed in pieces that assemble through hydrogen bonding +to the catalytic intron, in a manner analogous to the assembly of the +snRNAs in the spliceosome. + + +The similarities in mechanism have led to the suggestion that the +spliceosome-catalyzed splicing of mRNA precursors evolved from RNAcatalyzed self-splicing. Group II splicing may well be an intermediate +between group I splicing and the splicing in the nuclei of higher +eukaryotes. A major step in this transition was the transfer of catalytic +power from the intron itself to other molecules. The formation of +spliceosomes gave genes a new freedom because introns were no longer +constrained to provide the catalytic center for splicing. Another +advantage of external catalysts for splicing is that they can be more +readily regulated. + + +However, it is important to note that similarities do not establish +ancestry. The similarities between group II introns and mRNA splicing +may be a result of convergent evolution. Perhaps there are only a +limited number of ways to carry out efficient, specific intron excision. +The determination of whether these similarities stem from ancestry or +from chemistry will require expanding our understanding of RNA +biochemistry. + + +###### **Self–Check Question** + +Compare and contrast the three different splicing mechanisms that have been identified. + +#### **RNA enzymes can promote many reactions,** **including RNA polymerization** + + +In this chapter, we have discussed the role of many different RNA +molecules, including those that promote reactions such as splicing. In +Chapter 30, we will discuss the key roles that RNAs play in protein +biosynthesis, and we will see that ribosomal RNAs have a significant +role in the catalysis of peptide bonds. + + +While the ribosomal RNAs have a role in the polymerization of amino +acids into proteins, some RNAs are also capable of catalyzing the +polymerization of other RNA molecules or even themselves. In vitro +experiments have identified the possibility of self-ligating ribozymes: +RNA molecules capable of joining other, short RNAs to their own end. +The ability of RNAs to direct the synthesis of other catalytic molecules +gives us a glimpse into how, in a primordial RNA world, ribozymes may +have accelerated chemical reactions critical for life. + + +### **Chapter 29 Summary** + +**29.1 RNA Molecules Play Different Roles, Primarily in Gene** +**Expression** + + +RNA molecules are integral to many different cellular functions; +for example, protein biosynthesis. +Some RNAs can direct their own modification. + + +**29.2 RNA Polymerases Catalyze Transcription** + + +RNA polymerases synthesize all cellular RNA molecules +according to instructions given by DNA templates. +The direction of RNA synthesis is, as in DNA synthesis. + + +RNA polymerases, unlike DNA polymerases, do not need a +primer. +RNA polymerase in _E. coli_ is a multisubunit enzyme. The +subunit composition of the holoenzyme is + + +. +and that of the core enzyme is + + +Transcription is initiated at promoter sites. +The σ subunit enables the holoenzyme to recognize promoter +sites. The σ subunit usually dissociates from the holoenzyme +after the initiation of the new chain. +Elongation takes place at transcription bubbles that move along +the DNA template at a rate of about 50 nucleotides per second. +The nascent RNA chain contains stop signals that end +transcription. One stop signal is an RNA hairpin, which is +followed by several U residues. A different stop signal is read by +the _rho_ protein, an ATPase. +In _E. coli,_ precursors of transfer RNA and ribosomal RNA are +cleaved and chemically modified after transcription, whereas + + +messenger RNA is used unchanged as a template for protein +synthesis. + + +**29.3 Transcription Is Highly Regulated** + + +Bacteria use alternate σ factors to adjust the transcription levels +of various genes in response to external stimuli. +Some genes are regulated by riboswitches, structures that form +in RNA transcripts and bind specific metabolites. +Eukaryotic DNA is tightly bound to basic proteins called +histones; the combination is called chromatin. DNA wraps +around an octamer of core histones to form a nucleosome. +There are three types of eukaryotic RNA polymerases in the +nucleus, where transcription takes place: RNA polymerase I +makes ribosomal RNA precursors, II makes messenger RNA +precursors, and III makes transfer RNA precursors. +Eukaryotic promoters are composed of several different +elements. The activity of many promoters is greatly increased +by enhancer sequences that have no promoter activity of their + +own. + + +**29.4 Some RNA Transcription Products Are Processed** + + +The ends of mRNA precursors become capped and + + +methylated during transcription. +A +poly(A) tail is added to most mRNA precursors after the + + +nascent chain has been cleaved by an endonuclease. +The splicing of mRNA precursors is carried out by +spliceosomes, which consist of small nuclear +ribonucleoproteins. Splice sites in mRNA precursors are +specified by sequences at ends of introns and by branch sites +near the ends of introns. + + +RNA editing alters the nucleotide sequence of some mRNAs, +such as the one for apolipoprotein B. + + +**29.5 The Discovery of Catalytic RNA Revealed a Unique Splicing** +**Mechanism** + + +Some RNA molecules undergo self-splicing in the absence of +protein. +Spliceosome-catalyzed splicing may have evolved from selfsplicing. +The discovery of catalytic RNA has opened new vistas in our +exploration of early stages of molecular evolution and the +origins of life. + +### **Key Terms** + + +transcription +RNA polymerase +promoter +transcription bubble +sigma ( σ ) subunit + +consensus sequence +rho ( ρ ) protein +riboswitch + +chromatin + +nucleosome + + carboxyl terminal domain (CTD) +TATA box +transcription factor +enhancer +small nucleolar ribonucleoprotein (snoRNP) + + pre mRNA + + +cap + + +poly(A) tail +RNA splicing +spliceosome +small nuclear RNA (snRNA) +small nuclear ribonucleoprotein (snRNP) +alternative splicing +microRNA + +RNA editing +ribozyme + + self splicing introns + +### **Problems** + + +**1.** Why is RNA synthesis not as carefully monitored for errors +as is DNA synthesis? 1, 2 + + +**2.** What are the functions of RNA polymerases? Select all that +apply. 1, 3 + + +a. Polymerization of polypeptides from RNA transcripts +b. Elongation of ribosomal RNA (rRNA) + +c. Initiation of transcription at promoter sites +d. Elongation of messenger RNA (mRNA) transcripts + +e. Initiation of translation from RNA transcripts + + +**3.** The overall structures of RNA polymerase and DNA +polymerase are very different, yet their active sites show +considerable similarities. What do the similarities suggest +about the evolutionary relationship between these two +important enzymes? 1 + + +**4.** The sequence of part of an mRNA transcript is + + +What is the sequence of the DNA coding strand? Of the DNA +template strand? 1 + + +**5.** Sigma protein by itself does not bind to promoter sites. +Predict the effect of a mutation enabling σ to bind to the +region in the absence of other subunits of RNA polymerase. + + +2 + + +**6.** The molecular weight of an amino acid is approximately 110 +Da, and _E. coli_ RNA polymerase has a transcription rate of +approximately 5050 nucleotides per second. What is the +minimum length of time required by _E.coli_ polymerase for the +synthesis of an mRNA encoding a 100-kDa protein? Round your +answer to the nearest whole number. 1 + + +**7.** The autoradiograph below depicts several bacterial genes +undergoing transcription. Identify the DNA. What are the +strands of increasing length? Where is the beginning of +transcription? The end of transcription? What can you +conclude about the number of enzymes participating in RNA +synthesis on a given gene? 2 + + +a. Splicing occurs while the mRNA is attached to the + +nucleosome. + +b. One mRNA can sometimes code for more than one + + +protein by splicing at alternative sites. +c. Splicing occurs while the mRNA is still in the nucleus. +d. In splicing, intron sequences are removed from the + +mRNA in the form of lariats (loops) and are degraded. +e. 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Discuss the primary function of RNA polymerases, the reaction they catalyze, and + +the chemical mechanism of that reaction. +2. Describe transcription, including the processes of initiation, elongation, and + +termination. +3. Compare the roles of eukaryotic RNA polymerases I, II, and III in producing + +ribosomal, transfer, and messenger RNAs. +4. Recognize the significance of transcription factors and enhancers in the regulation + +of transcription in eukaryotes. +5. Describe the process of RNA splicing, including the roles of the spliceosome and + +self-splicing RNA molecules. +6. Understand some of the differences between transcription in bacteria and in + +eukaryotes. + + +DNA stores genetic information in a stable form that can be readily +replicated. The expression of this genetic information requires its +flow from DNA to RNA and, usually, to protein, as was introduced in + +. +Chapter 8 This chapter examines transcription, which, you will +recall, is the process of synthesizing an RNA transcript from a DNA +template, transferring the sequence information within the DNA to +the new RNA molecule. We begin with a brief discussion of the +diverse types of RNA molecules; then we will turn to RNA +polymerases, the large and complex enzymes that carry out the + + +synthetic process. This will lead into a discussion of transcription in +bacteria and focus on the three stages of transcription: promoter +binding and initiation, elongation of the nascent RNA transcript, +and termination. We then examine transcription in eukaryotes, +focusing on the distinctions between bacterial and eukaryotic +transcription. + + +# **29.1 RNA Molecules Play Different** **Roles, Primarily in Gene Expression** + +While the function of some RNAs has been known for some time, +other classes of RNAs have only recently been discovered. The +investigation of some of these RNA molecules has been one of the +most productive areas of biochemical research in recent years. + +### **RNAs play key roles in protein** **biosynthesis** + + +As we will explore in Chapter 30, the long-known ribosomal and +transfer RNA molecules, along with messenger RNAs, are central to +protein synthesis. Ribosomal RNAs are critical components of +ribosomes (the sites of protein synthesis), and transfer RNAs play a +role in delivering amino acids (the building blocks of proteins) to the +ribosome. Messenger RNAs carry the information that ribosomes +use for the production of specific protein sequences. + +### **Some RNAs can guide modifications of** **themselves or other RNAs** + + +In eukaryotes, one of the most striking examples of RNA +modification is the splicing of mRNA precursors, a process that is +catalyzed by large complexes composed of both proteins and small +nuclear RNAs. These small nuclear RNAs play a crucial role in +guiding the splicing of messenger RNAs. + + +Remarkably, some RNA molecules can splice themselves in the +absence of other proteins and RNAs. This landmark discovery of +self-splicing introns, which we will discuss later in this chapter, +revealed that RNA molecules can serve as catalysts, which greatly +influenced our view of molecular evolution. Many other types of +RNAs, such as small regulatory RNAs and long noncoding RNAs, +have been discovered more recently, and while their functions are +still under active investigation, our understanding is rapidly +expanding. + +### **Some viruses have RNA genomes** + + +While DNA is the genetic material in most organisms, some viruses +have genomes made of RNA (Section 8.4). RNA viruses are +responsible for several diseases, such as influenza, polio, mumps, +Ebola, the common cold, and — of particular note — COVID-19. The +coronavirus SARS-CoV-2, which causes COVID-19, belongs to a +family of coronaviruses that have an unusually large, singlestranded RNA genome. Coronaviruses cause a variety of diseases in +mammals and birds that have a wide range of symptoms, including +respiratory distress in humans that can potentially be lethal. + + +RNA viruses vary in their genome organization. They have either +single- or double-stranded RNA molecules arranged in single or +multiple fragments. Inside their hosts, RNA viruses replicate their +genomes with a virus-encoded RNA polymerase that uses RNA as a +template. In some viruses, a complementary RNA molecule is made +from the single-stranded viral RNA genome, while in other viruses, a +double-stranded DNA copy is made that can integrate itself into the +host’s genome. Viral RNA polymerases do not have the same +proofreading ability of other polymerases, which leads to high +mutation rates of RNA viruses. + + +### **Messenger RNA vaccines provide** **protection against diseases** + +Messenger RNA (mRNA) vaccines take advantage of the fact that +cells can be tricked into making proteins they don’t usually make, +even those from other organisms such as viruses. Unlike other +vaccines that use parts of a weakened or inactivated pathogen to +trigger an immune response, mRNA vaccines — including those +available for the SARS-CoV-2 virus — use sections of pathogenic +(usually viral) mRNA that have been generated in the laboratory. +When human cells are injected with this mRNA, they will produce +the corresponding protein from this set of instructions. This +“foreign” protein will be presented on the surface of the cells, +trigger an immune response, and provide some level of protection if +and when the actual pathogen invades. Even though research on +mRNA vaccines had been going on for many years, the COVID-19 +pandemic accelerated the development, approval, and distribution +of mRNA vaccines to the public. + +##### **Self–Check Question** + + +Compare the biological roles of RNA and DNA. What aspects of the structure and +chemistry of RNA make it so versatile? + + +## **29.2 RNA Polymerases Catalyze** **Transcription** + +**Transcription**, the synthesis of RNA molecules from a DNA template, is +catalyzed by large enzymes called **RNA polymerases** . The basic +biochemistry of RNA synthesis is shared by all organisms, a +commonality that has been beautifully illustrated by the threedimensional structures of representative RNA polymerases from +prokaryotes and eukaryotes ( **Figure 29.1** ). Despite substantial +differences in size and number of polypeptide subunits, the overall +structures of these enzymes are quite similar, revealing a common +evolutionary origin. + + +RNA polymerases are very large, complex enzymes. For example, the +core of the RNA polymerase of _E. coli_ consists of five kinds of subunits + + +with the composition ( **Table 29.1** ). A typical eukaryotic RNA + + +polymerase is larger and more complex, having 12 subunits and a total +molecular mass of more than 500 kDa. Despite this complexity, the +detailed structures of RNA polymerases have been determined by x-ray +crystallography in work pioneered by Roger Kornberg and Seth Darst. +The structures of many additional RNA polymerase complexes have +been determined by cryo-electron microscopy. + + +**TABLE 29.1 Subunits of RNA polymerase from** _**E. coli**_ + + +α _rpoA_ 2 37 + + +β _rpoB_ 1 151 + + +_rpoC_ 1 155 + + +ω _rpoZ_ 1 10 + + +_rpoD_ 1 70 + + +#### **RNA synthesis comprises three stages:** **initiation, elongation, and termination** + +RNA synthesis, like all biological polymerization reactions, takes place +in three stages: _initiation_, _elongation_, and _termination_ . RNA polymerases +perform multiple functions in this process: + + +or +1. They search DNA for initiation sites, also called _promoter sites_ + +simply **promoters** . For instance, _E. coli_ DNA has about 2000 + + +promoters in its genome. + + +2. They unwind a short stretch of double-helical DNA to produce + +single-stranded DNA templates from which the sequence of bases +can be easily read out. + + +3. They select the correct ribonucleoside triphosphate and catalyze + +the formation of a phosphodiester bond. This process is repeated +many times as the enzyme moves along the DNA template. RNA +polymerase is completely processive — a transcript is synthesized +from start to end by a single RNA polymerase molecule. + + +4. They detect termination signals that specify where a transcript + +ends. + + +5. Their activity is regulated by activator and repressor proteins that + +interact with the promoter and modulate the ability of the RNA +polymerase to initiate transcription. Gene expression is controlled +substantially at the level of transcription, as will be discussed in +detail in . +Chapter 31 + + +The chemistry of RNA synthesis is identical for all forms of RNA, +including messenger RNAs, transfer RNAs, ribosomal RNAs, and small +regulatory RNAs, so the basic steps just outlined apply to all forms. +Their synthetic processes differ mainly in regulation, the specific RNA +polymerase that creates them, and their posttranscriptional processing. + + +#### **RNA polymerases catalyze the formation of a** **phosphodiester bond** + +The fundamental reaction of RNA synthesis, like that of DNA synthesis, +is the formation of a phosphodiester bond. The -hydroxyl group of the + + + last nucleotide in the chain makes a nucleophilic attack on the α +phosphoryl group of the incoming nucleoside triphosphate, releasing a +pyrophosphate. + + +The catalytic sites of all RNA polymerases include two metal ions, +normally magnesium ions ( **Figure 29.2** ). One ion remains tightly bound +to the enzyme, whereas the other ion comes in with the nucleoside +triphosphate and leaves with the pyrophosphate. Three conserved +aspartate residues participate in binding these metal ions. Given the +recent appreciation of the role of a third metal ion in the active site of +DNA polymerases (see Section 28.1), it will be interesting to see if RNA +polymerases show similarities to that model. + + +The polymerization reactions that are catalyzed by both prokaryotic and +eukaryotic RNA polymerases take place within a complex in DNA +termed a **transcription bubble** ( **Figure 29.3** ). This complex consists of +double-stranded DNA that has been locally unwound in a region of +approximately 17 base pairs. The edges of the bases that normally take +part in Watson–Crick base pairs are exposed in the unwound region. We +will begin with a detailed examination of the elongation process, +including the role of the DNA template read by RNA polymerase and the +reactions catalyzed by the polymerase, before returning to the more +complex processes of initiation and termination. + + +#### **RNA chains are formed de novo and grow in** **the -to- direction** + +Let us begin our examination of transcription by considering the DNA +template. The first nucleotide (the start site) of a DNA sequence to be +transcribed is denoted as and the second one as ; the nucleotide + +preceding the start site is denoted as . These designations refer to the + + +coding strand of DNA. Recall that the sequence of the _template strand_ of +DNA is the complement of that of the RNA transcript ( **Figure 29.4** ). In +contrast, the _coding strand_ of DNA has the same sequence as that of the +RNA transcript except for thymine (T) in place of uracil (U). The coding + +strand is also known as the _sense_ _strand_, and the template strand as + + +the _antisense_ _strand_ . + + + + +In contrast with DNA synthesis, RNA synthesis can start de novo, +without the requirement for a primer. Most newly synthesized RNA +chains carry a highly distinctive tag on the end: the first base at that + + +end is either pppG or pppA. + + +The presence of the triphosphate moiety confirms that RNA synthesis +starts at the end. + + +The dinucleotide shown above is synthesized by RNA polymerase as +part of the complex process of initiation, which will be discussed later +in the chapter. After initiation takes place, RNA polymerase elongates +the nucleic acid chain as follows ( **Figure 29.5** ). + + +1. A ribonucleoside triphosphate binds in the active site of the RNA + +polymerase directly adjacent to the growing RNA chain, and it +forms a Watson–Crick base pair with the template strand. + + +2. The -hydroxyl group of the growing RNA chain, which is oriented + + + and activated by the tightly bound metal ion, attacks the α +phosphoryl group to form a new phosphodiester bond, displacing +pyrophosphate. + + +3. Next, the RNA–DNA hybrid must move relative to the polymerase to + + +bring the end of the newly added nucleotide into proper position + + +for the next nucleotide to be added. This translocation step does not +include breaking any bonds between base pairs and is reversible; +but, once it has taken place, the addition of the next nucleotide, +favored by the triphosphate cleavage and pyrophosphate release +and cleavage, drives the polymerization reaction forward. + + +The lengths of the RNA–DNA hybrid and of the unwound region of DNA +stay rather constant as RNA polymerase moves along the DNA template. +The length of the RNA–DNA hybrid is determined by a structure within +the enzyme that forces the RNA–DNA hybrid to separate, allowing the +RNA chain to exit from the enzyme and the DNA chain to rejoin its DNA +partner ( **Figure 29.6** ). + + +#### **RNA polymerases backtrack and correct** **errors** + +The RNA–DNA hybrid can also move in the direction opposite that of +elongation ( **Figure 29.7** ). This backtracking is energetically less +favorable than moving forward because it breaks the bonds between a +base pair. However, backtracking is very important for _proofreading_ . The +incorporation of an incorrect nucleotide introduces a non-Watson–Crick +base pair. In this case, breaking the bonds between this base pair and +backtracking is energetically less costly. + + + + + +After the polymerase has backtracked, the phosphodiester bond one +base pair before the one that has just formed is adjacent to the metal ion +in the active site. In this position, a hydrolysis reaction in which a water +molecule attacks the phosphate can result in the cleavage of the + + +phosphodiester bond and the release of a dinucleotide that includes the +incorrect nucleotide. + + +Studies of single molecules of RNA polymerase have confirmed that the +enzymes pause and backtrack to correct errors. Furthermore, these +proofreading activities are often enhanced by accessory proteins. + + +or +The final error rate of the order of one mistake per + + +nucleotides is higher than that for DNA replication, including all errorcorrecting mechanisms. The lower fidelity of RNA synthesis can be +tolerated because mistakes are not transmitted to progeny. For most +genes, many RNA transcripts are synthesized; a few defective +transcripts are unlikely to be harmful. + +###### **Self–Check Question** + + +List some ways in which RNA polymerases are similar to DNA polymerases, and how they are +different. + +#### **RNA polymerase binds to promoter sites on** **the DNA template in bacteria to initiate** **transcription** + + +While the elongation process is common to all organisms, the processes +of initiation and termination differ substantially in bacteria and +eukaryotes. We begin with a discussion of these processes in bacteria, +starting with initiation of transcription. + + +The bacterial RNA polymerase discussed earlier with the composition + + +is referred to as the . The inclusion of an additional +_core enzyme_ + + +subunit ( σ ) produces the _holoenzyme_ with composition . The **σ** + +**subunit** helps find the promoters, sites on DNA where transcription +begins. At these sites, the σ subunit participates in the initiation of RNA +synthesis and then dissociates from the rest of the enzyme. + + +Sequences upstream of the promoter site are important in determining +where transcription begins. A striking pattern is evident when the +sequences of bacterial promoters are compared: Two common motifs +are present on the upstream side of the transcription start site. They are + +known as the and the +_sequence_ _sequence_ because they are +centered at about 10 and 35 nucleotides upstream of the start site. The +region containing these sequences is called the _core promoter_ . The + +and sequences are each 6 bp long. Their **consensus sequences**, +deduced from analyses of many promoters ( **Figure 29.8** ), are: + + +Promoters differ markedly in their efficacy. Some genes are transcribed +frequently — as often as every 2 seconds in _E. coli_ . The promoters for +these genes are referred to as _strong promoters_ . In contrast, other genes +are transcribed much less frequently, about once in 10 minutes; the +promoters for these genes are _weak promoters_ . The and +regions of most strong promoters have sequences that correspond +closely to the consensus sequences, whereas weak promoters tend to +have multiple substitutions at these sites. Indeed, mutation of a single +base in either the sequence or the sequence can diminish +promoter activity. + + +The distance between these conserved sequences also is important; a +separation of 17 nucleotides is optimal. Thus, the efficiency or strength +of a promoter sequence serves to regulate transcription. Regulatory +proteins that bind to specific sequences near promoter sites and +interact with RNA polymerase (Chapter 31) also markedly influence the +frequency of transcription of many genes. + + +Outside the core promoter in a subset of highly expressed genes is the +_upstream element_ (also called the UP element). This sequence is present +from 40 to 60 nucleotides upstream of the transcription start site. The +UP element is bound by the α subunit of RNA polymerase and serves to +increase the efficiency of transcription by creating an additional +interaction site for the polymerase. + +#### **Sigma subunits of RNA polymerase in** **bacteria recognize promoter sites** + + +To initiate transcription, the core of RNA polymerase must bind + + +the promoter. However, it is the σ subunit that makes this binding +possible by enabling RNA polymerase to recognize promoter sites. In +the presence of the σ subunit, the RNA polymerase binds weakly to the +DNA and slides along the double helix until it dissociates or encounters +a promoter. The σ subunit recognizes the promoter through several +interactions with the nucleotide bases of the promoter DNA. The +structure of a bacterial RNA polymerase holoenzyme bound to a +promoter site shows the σ subunit interacting with DNA at the and + + +regions essential to promoter recognition ( **Figure 29.9** ). Therefore, + +the σ +subunit is responsible for the specific binding of the RNA +polymerase to a promoter site on the template DNA. The σ subunit is +generally released when the nascent RNA chain reaches 9 or 10 +nucleotides in length. After its release, it can associate with another +core enzyme and assist in a new round of initiation. + + +#### **The template double helix must be unwound** **for transcription to take place** + +Although the bacterial RNA polymerase can search for promoter sites +when bound to double-helical DNA, a segment of the DNA double helix +must be unwound before synthesis can begin. The transition from the +closed promoter complex (in which DNA is double helical) to the open +promoter complex (in which a DNA segment is unwound) is an essential +event in both bacterial and eukaryotic transcription. In bacteria it is the +RNA polymerase itself that accomplishes this ( **Figure 29.10** ), while in +eukaryotes additional proteins are required to unwind the DNA +template. + + + + + +We know that, in bacteria, the free energy necessary to break the bonds +between approximately 17 base pairs in the double helix is derived from +additional interactions between the template and the bacterial RNA +polymerase. These interactions become possible when the DNA distorts +to wrap around the RNA polymerase; they also occur between the +single-stranded DNA regions and other parts of the enzyme. These +interactions stabilize the open promoter complex and help pull the +template strand into the active site. The element remains in a + + +double-helical state, whereas the element is unwound. The stage is + + +now set for the formation of the first phosphodiester bond of the new +RNA chain. + +#### **Elongation takes place at transcription** **bubbles that move along the DNA template** + + +The elongation phase of RNA synthesis begins with the formation of the +first phosphodiester bond, after which repeated cycles of nucleotide +addition can take place. However, until about 10 nucleotides have been +added, RNA polymerase sometimes releases the short RNA, which +dissociates from the DNA and gets degraded. Once RNA polymerase +passes this point, the enzyme stays bound to its template until a +termination signal is reached. + + +The region containing the unwound DNA template and nascent RNA +corresponds to the transcription bubble ( **Figure 29.11** ). The newly +synthesized RNA forms a hybrid helix with the template DNA strand. +This RNA–DNA helix is about 8 bp long, which corresponds to nearly + + +one turn of a double helix. The -hydroxyl group of the RNA in this + + +hybrid helix is positioned so that it can attack the α -phosphorus atom of +an incoming ribonucleoside triphosphate. The core bacterial RNA +polymerase also contains a binding site for the coding strand of DNA. + + +As in the initiation phase, about 17 bp of DNA are unwound throughout +the elongation phase. The transcription bubble moves a distance of 170 +Å (17 nm) in a second, which corresponds to a rate of elongation of +about 50 nucleotides per second. Although rapid, it is much slower than +the rate of DNA synthesis, which is 800 nucleotides per second. + +#### **Sequences within the newly transcribed RNA** **signal termination** + + +How does the RNA polymerase know where to stop transcription? In the +termination phase of transcription, the formation of phosphodiester +bonds ceases, the RNA–DNA hybrid dissociates, the unwound region of +DNA rewinds, and RNA polymerase releases the DNA. This process is as +precisely controlled as initiation. So what determines where + + +transcription is terminated? In both eukaryotes and bacteria, the +transcribed regions of DNA templates contain so-called _intrinsic_ +termination signals _._ + + +In bacteria, the simplest intrinsic termination signal is a palindromic +GC-rich region followed by an AT-rich region. The RNA transcript of this +DNA palindrome is self-complementary ( **Figure 29.12** ). Hence, its bases +can pair to form a hairpin structure with a stem and loop, a structure +favored by its high content of G and C residues. Guanine–cytosine base +pairs are more stable than adenine–thymine pairs, primarily because of +the preferred base-stacking interactions in G–C base pairs (Section 1.3). +This stable hairpin is followed by a sequence of four or more uracil +residues, which also are crucial for termination. The RNA transcript +ends within or just after them. + + +How does this combination hairpin–oligo(U) structure terminate +transcription? First, RNA polymerase likely pauses immediately after it +has synthesized a stretch of RNA that folds into a hairpin. Furthermore, +the RNA–DNA hybrid helix produced after the hairpin is unstable +because its rU–dA base pairs are the weakest of the four kinds. Hence, +the pause in transcription caused by the hairpin permits the weakly +bound nascent RNA to dissociate from the DNA template and then from +the enzyme. The solitary DNA template strand rejoins its partner to reform the DNA duplex, and the transcription bubble closes. + +#### **In bacteria, the rho protein helps to** **terminate the transcription of some genes** + + +Bacterial RNA polymerase needs no help to terminate transcription at +the intrinsic sites described above. At other sites, however, termination +requires the participation of an additional factor. This discovery was +prompted by the observation that some RNA molecules synthesized in +vitro by RNA polymerase acting alone are longer than those made in +vivo. The missing factor, a protein that caused the correct termination, +was isolated and named **rho (** **ρ** **)** . + + +Additional information about the action of ρ was obtained by adding +this termination factor to an incubation mixture at various times after +the initiation of RNA synthesis ( **Figure 29.13** ). RNAs with sedimentation +coefficients of 10S, 13S, and 17S were obtained when ρ was added at +initiation, a few seconds after initiation, and 2 minutes after initiation, +respectively. If no ρ was added, transcription yielded a 23S RNA product. +It is evident that the template contains at least three termination sites +that respond to ρ (yielding 10S, 13S, and 17S RNA) and one termination +site that does not (yielding 23S RNA). Thus, specific termination at a site +producing 23S RNA can take place in the absence of ρ . However, ρ +detects additional termination signals that are not recognized by RNA +polymerase alone. + + +The ρ protein promotes about 20% of termination events in bacteria, +but exactly how it selects its target termination signals is not clear. +Unlike the hairpin–oligo(U) sequence of intrinsic termination sites, the +identification of conserved patterns in ρ -dependent terminators has +proven more difficult. + + +How does ρ promote the termination of RNA synthesis? A key clue is the +finding that ρ is hexameric and hydrolyzes ATP in the presence of +single-stranded RNA but not in the presence of DNA or duplex RNA. +Thus ρ is a helicase, homologous to the hexameric helicases that we +encountered in our discussion of DNA replication (Section 28.1). The +role of ρ in the termination of transcription in bacteria is as follows +( **Figure 29.14** ): + + +The ρ protein is brought into action by sequences located in the +nascent RNA that are rich in cytosine and poor in guanine. + + +A stretch of nucleotides is bound in such a way that the RNA passes +through the center of the structure. + + +The helicase activity of ρ enables the protein to pull the nascent +RNA while pursuing RNA polymerase. + + +When ρ catches RNA polymerase at the transcription bubble, it +breaks the RNA–DNA hybrid by functioning as an RNA–DNA +helicase. + + + + + +Proteins in addition to ρ may promote termination. For example, the +NusA protein enables RNA polymerase in _E. coli_ to recognize a +characteristic class of termination sites. A common feature of + +transcription termination, whether it relies on a protein or not, is that + + +the functioning signals lie in newly synthesized RNA rather than in the + +_._ +DNA template + + +## **29.3 Transcription Is Highly** **Regulated** + +As we will see in Chapter 31, the level at which different genes are +transcribed is highly regulated. Regulated gene expression is critical for +the development of multicellular organisms, the differentiation of +various cell types, and the response of bacteria to changes in their +environment. Here, we will discuss a few examples of how transcription +can be controlled. + +#### **Alternative sigma subunits in bacteria** **control transcription in response to changes** **in conditions** + + +As noted above, the σ factor allows for the specific binding of the +bacterial RNA polymerase to a promoter site on the template DNA. _E._ +_coli_ has seven distinct σ factors for recognizing several types of +promoter sequences in _E. coli_ DNA. The type that recognizes the + + +consensus sequences described earlier is called because it has a + + +mass of 70 kDa. A different σ factor comes into play when the + + +temperature is raised abruptly. _E. coli_ responds by synthesizing, + + +which recognizes the promoters of so-called _heat-shock genes_ . These +promoters exhibit sequences that are somewhat different from the + +sequence for standard promoters ( **Figure 29.15** ). The increased +transcription of heat-shock genes leads to the coordinated synthesis of a +series of protective proteins. Other σ factors respond to environmental +conditions, such as nitrogen starvation. These findings demonstrate + + +that σ plays the key role in determining when and where RNA +polymerase initiates transcription. + + + +Some other bacteria contain a much larger number of σ factors. For +example, the genome of the soil bacterium _Streptomyces coelicolor_ +encodes more than 60 σ factors recognized on the basis of their amino +acid sequences. This repertoire allows these cells to adjust their geneexpression programs to the wide range of conditions, with regard to +nutrients and competing organisms, that they may experience. + +#### **Some messenger RNAs directly sense** **metabolite concentrations** + + +As we shall explore in Chapter 31, the expression of many genes is +controlled in response to the concentrations of metabolites and +signaling molecules within cells. One set of control mechanisms found +in both prokaryotes and eukaryotes depends on the remarkable ability +of some mRNA molecules to form secondary structures that are capable +of directly binding small molecules. These structures are termed +**riboswitches** . + + +Consider a riboswitch that controls the synthesis of genes that +participate in the biosynthesis of riboflavin in the bacterium _Bacillus_ +_subtilis_ ( **Figure 29.16** ). When flavin mononucleotide (FMN), a key +intermediate in riboflavin biosynthesis, is present at high +concentration, it binds to the RNA transcript. Binding of FMN to the +transcript induces a hairpin structure that favors premature +termination. By trapping the RNA transcript in this terminationfavoring conformation, FMN prevents the production of functional fulllength mRNA. However, when FMN is present at low concentration, it +does not readily bind to the mRNA. Without FMN bound, the transcript +adopts an alternative conformation without the terminator hairpin, +allowing the production of the full-length mRNA. The occurrence of +riboswitches serves as a vivid illustration of how RNAs are capable of +forming elaborate, functional structures, though in the absence of +specific information we tend to depict them as simple lines. + + + + +#### **Control of transcription in eukaryotes is** **highly complex** + +We turn now to transcription in eukaryotes, a much more complex +process than in bacteria. Eukaryotic cells have a remarkable ability to +regulate precisely the time at which each gene is transcribed and how +much RNA is produced. This ability led to the evolution of multicellular +eukaryotes with distinct tissues. That is, multicellular eukaryotes use +differential transcriptional regulation to create different cell types. + + +Gene expression is influenced by three important characteristics unique +to eukaryotes: the nuclear membrane, complex transcriptional +regulation, and RNA processing. + + +1. _The nuclear membrane allows transcription and translation to take place_ + +_in different cellular compartments._ Transcription takes place in the +membrane-bound nucleus, whereas translation takes place outside +the nucleus in the cytoplasm. In bacteria, the two processes are +closely coupled ( **Figure 29.17** ). Indeed, the translation of bacterial +mRNA begins while the transcript is still being synthesized. The +spatial and temporal separation of transcription and translation +enables eukaryotes to regulate gene expression in much more +intricate ways, contributing to the richness of eukaryotic form and +function. + + +2. _A variety of types of promoter elements enables complex transcriptional_ + +_regulation_ . Like bacteria, eukaryotes rely on conserved sequences +in DNA to regulate the initiation of transcription. But bacteria have +only three promoter elements (the, and UP elements), +whereas eukaryotes use a variety of types of promoter elements, +each identified by its own conserved sequence. Not all possible +types will be present together in the same promoter. In eukaryotes, +elements that regulate transcription can be found upstream or +downstream of the start site and sometimes at distances much +farther from the start site than in prokaryotes. For example, +enhancer elements located on DNA far from the start site increase +the promoter activity of specific genes. + + +3. _The degree of RNA processing is much greater in eukaryotes than in_ + +_bacteria_ . Although both bacteria and eukaryotes modify RNA, +eukaryotes very extensively process nascent RNA destined to +become mRNA. This processing includes modifications to both +ends and, most significantly, splicing out segments of the primary +transcript. RNA processing is described in Section 29.4. + +#### **Eukaryotic DNA is organized into chromatin** + + +Whereas bacterial genomic DNA is relatively accessible to the proteins +involved in transcription, eukaryotic DNA is packaged into **chromatin**, a +complex formed between the DNA and a particular set of proteins. +Chromatin compacts and organizes eukaryotic DNA, and its presence +has dramatic consequences for gene regulation. Although the principles +for the construction of chromatin are relatively simple, the chromatin +structure for a complete genome is quite complicated. Importantly, in +any given eukaryotic cell, some genes and their associated regulatory +regions are relatively accessible for transcription and regulation, +whereas other genes are tightly packaged, less accessible, and therefore +inactive. Eukaryotic gene regulation frequently requires the +manipulation of chromatin structure. + + +Chromatin viewed with the electron microscope has the appearance of +beads on a string ( **Figure 29.18** ). Partial digestion of chromatin with +DNase exposes these particles, which consist of fragments of DNA (the +“string”) wrapped around octamers of proteins called _histones_ (the +“beads”). The complex formed by a histone octamer and a 145-bp DNA +fragment is called the **nucleosome** ( **Figure 29.19** ). + + +**FIGURE 29.18 Eukaryotic chromatin structure resembles beads on a string.** In this +electron micrograph of chromatin, the “beads” correspond to DNA complexed with +specific proteins into nucleosomes. Each bead has a diameter of approximately 100 +Å. + + +The overall structure of the nucleosome was revealed through electron +microscopic and x-ray crystallographic studies pioneered by Aaron Klug +and his colleagues. More recently, the three-dimensional structures of +reconstituted nucleosomes have been determined to higher resolution +by x-ray diffraction methods. The histone octamer is a complex of four +different types of histones (H2A, H2B, H3, and H4) that are homologous +and similar in structure. + + +The eight histones in the core are arranged into a tetramer + + +and a pair of H2A–H2B dimers. The tetramer and dimers come together +to form a left-handed superhelical ramp around which the DNA wraps. +In addition, each histone has an amino-terminal tail that extends out +from the core structure. These tails are flexible and contain many lysine +and arginine residues. As we shall see in Chapter 31, covalent +modifications of these tails play an essential role in regulating gene +expression. + + +#### **Three types of RNA polymerase synthesize** **RNA in eukaryotic cells** + +In bacteria, RNA is synthesized by a single kind of polymerase. In +contrast, the nucleus of a typical eukaryotic cell contains three types of +RNA polymerase differing in template specificity and location in the +nucleus ( **Table 29.2** ). The three polymerases are named for the order in +which they were discovered, which has no bearing on the relative +importance of their function. We will discuss them in an order that +reflects their similarities in localization, function, and regulation. We +will emphasize RNA polymerase II, since it transcribes all of the +protein-coding genes and has therefore been the focus of much +research investigating transcriptional mechanisms. + + +**TABLE 29.2 Eukaryotic RNA polymerases** + + + + + + + +I Nucleolus 18S, 5.8S, and 28S rRNA Insensitive + + + +II Nucleoplas + +m + + +III Nucleoplas + +m + + + +mRNA precursors and snRNA Strongly inhibited + + +tRNA and 5S rRNA Inhibited by high concentrations + + + +_RNA polymerase I_ is located in specialized structures within the nucleus +called nucleoli, where it transcribes the tandem array of genes for 18S, +5.8S, and 28S rRNA. The other rRNA molecule (5S rRNA) and all the +tRNA molecules are synthesized by _RNA polymerase III_, which is located +in the nucleoplasm rather than in nucleoli. _RNA polymerase II_, which + + +also is located in the nucleoplasm, synthesizes the precursors of mRNA +as well as several small RNA molecules, such as those of the splicing +apparatus and many of the precursors to small regulatory RNAs. All +three of the polymerases are large proteins, containing from 8 to 14 +subunits and having total molecular masses greater than 500 kDa (or 0.5 +MDa), and it is likely that they evolved from a single enzyme that was +present in a common ancestor of eukaryotes, bacteria, and archaea. In +fact, many components of the eukaryotic transcriptional machinery +evolved from those in a common ancestor. + + +Although all eukaryotic RNA polymerases are homologous to one +another and to prokaryotic RNA polymerases, RNA polymerase II + +**-** +contains a unique **carboxyl** **terminal domain** (CTD) on the 220-kDa +subunit; this domain is unusual because it contains multiple repeats of +a YSPTSPS consensus sequence. The activity of RNA polymerase II is +regulated by phosphorylation, mainly on the serine residues of the CTD. + + +The different polymerases were originally distinguished through their +variable responses to the toxin α -amanitin, a cyclic octapeptide that +contains several modified amino acids and is produced by a genus of +poisonous mushroom ( **Figure 29.20** ). α -Amanitin binds very tightly + + +to RNA polymerase II and thereby blocks the elongation + + +phase of RNA synthesis. Higher concentrations of α -amanitin (1 μ M) +inhibit RNA polymerase III, whereas RNA polymerase I is insensitive to +this toxin. This pattern of sensitivity is highly conserved throughout the +animal and plant kingdoms. + + +**FIGURE 29.20** **α** **-Amanitin is produced by poisonous mushrooms in the genus** +_**Amanita**_ **.** Pictured is _Amanita phalloides_, also called the _death cap_ or the _destroying_ +_angel_ . + + +Finally, eukaryotic polymerases differ from each other in the promoters +to which they bind. Eukaryotic genes, like prokaryotic genes, require +promoters for transcription initiation. Like prokaryotic promoters, +eukaryotic promoters consist of conserved sequences that attract the +polymerase to the start site. However, eukaryotic promoters differ +distinctly in sequence and position, depending on the type of RNA +polymerase that binds to them ( **Figure 29.21** ). + + +_The promoter sequences for RNA polymerase I are located in stretches of_ +_DNA separating the ribosomal DNA (rDNA) it transcribes._ These rRNA +genes are arranged in several hundred tandem repeats, each +containing a copy of each of three rRNA genes. At the +transcriptional start site lies a TATA-like sequence called the +_ribosomal initiator element_ (rInr). Farther upstream, 150 to 200 bp +from the start site, is the _upstream promoter element_ (UPE). Both +elements aid transcription by binding proteins that recruit RNA +polymerase I. + + +_Promoters for RNA polymerase II, like prokaryotic promoters, include a_ +_set of consensus sequences that define the start site and recruit the_ +_polymerase._ However, the promoter can contain any combination of +a number of possible consensus sequences. Unique to eukaryotes, +they also include enhancer elements that can be more than 1 kb +from the start site. + + +_Promoters for RNA polymerase III are within the transcribed sequence,_ +_downstream of the start site_ . This is contrast to promoters for RNA +polymerase I and II, which are _upstream_ of the transcription start +site. There are two types of intergenic promoters for RNA +polymerase III. Type I promoters, found in the 5S rRNA gene, +contain two short, conserved sequences, the A block and the C +block. Type II promoters, found in tRNA genes, consist of two 11-bp +sequences, the A block and the B block, situated about 15 bp from +either end of the gene. + + +#### **Three common elements can be found in the** **RNA polymerase II promoter region** + +RNA polymerase II transcribes all of the protein-coding genes in +eukaryotic cells. Promoters for RNA polymerase II, like those for +bacterial polymerases, are generally located upstream of the start site +for transcription. Because these sequences are on the _same_ molecule of +DNA as the genes being transcribed, they are called _cis-acting elements._ + + +1. The most commonly recognized cis-acting element for genes + +transcribed by RNA polymerase II is called the **TATA box** on the +basis of its consensus sequence ( **Figure 29.22** ). The TATA box is +usually found between positions and . Note that the + +eukaryotic TATA box closely resembles the prokaryotic +sequence (TATAAT) but is farther from the start site. The mutation +of a single base in the TATA box markedly impairs promoter +activity. Thus, the precise sequence, not just a high content of AT +pairs, is essential. + + + + + +2. The TATA box is often paired with an _initiator element_ (Inr), a + +sequence found at the transcriptional start site, between positions + +and . This sequence defines the start site because the other + + +promoter elements are at variable distances from that site. Its +presence increases transcriptional activity. + + +3. A third element, the _downstream core promoter element_ (DPE), is + +commonly found in conjunction with the Inr in transcripts that +lack the TATA box. In contrast with the TATA box, the DPE is found +downstream of the start site, between positions and . + +#### **Regulatory cis-acting elements are** **recognized by different mechanisms** + + +Additional regulatory sequences are located between and . + + +Many promoters contain a _CAAT box_, and some contain a _GC box_ ( **Figure** +**29.23** ). Constitutive genes (genes that are continuously expressed rather +than regulated) tend to have GC boxes in their promoters. The positions +of these upstream sequences vary from one promoter to another, in +contrast with the quite constant location of the region in +prokaryotes. Another difference is that the CAAT box and the GC box +can be effective when present on the template (antisense) strand, unlike +the region, which must be present on the coding (sense) strand. + + +These differences between prokaryotes and eukaryotes correspond to +fundamentally different mechanisms for the recognition of cis-acting +elements. The and sequences in prokaryotic promoters are +binding sites for RNA polymerase and its associated σ factor. In +contrast, the TATA, CAAT, and GC boxes and other cis-acting elements in +eukaryotic promoters are recognized by proteins other than RNA +polymerase itself. + + +#### **The TFIID protein complex initiates the** **assembly of the active transcription complex** **in eukaryotes** + +Cis-acting elements constitute only part of the puzzle of eukaryotic gene +expression. **Transcription factors** that bind to these elements also are +required. For example, RNA polymerase II is guided to the start site by a +set of transcription factors known collectively as _TFII_ ( _TF_ stands for +transcription factor, and _II_ refers to RNA polymerase II). Individual TFII +factors are called TFIIA, TFIIB, and so on. + + +In TATA-box promoters, the key initial event is the recognition of the +TATA box by the TATA-box-binding protein (TBP), a 30-kDa component +of the 700-kDa TFIID complex. In TATA-less promoters, other proteins in +the TFIID complex bind the core promoter elements; however, because +less is known about these interactions, we will consider only the TATAbox–TBP binding interaction. TBP binds times as tightly to the TATA + + +box as to nonconsensus sequences; the dissociation constant of the +TBP–TATA-box complex is approximately 1 nM. + + +The TATA box of DNA binds to the concave surface of TBP, inducing +large conformational changes in the bound DNA ( **Figure 29.24** ). The +double helix is substantially unwound to widen its minor groove, +enabling it to make extensive contact with the antiparallel β strands on +the concave side of TBP. Hydrophobic interactions are prominent at this +interface. Four phenylalanine residues, for example, are intercalated +between base pairs of the TATA box. The flexibility of AT-rich sequences +is generally exploited here in bending the DNA. Immediately outside the +TATA box, classical B-DNA resumes. The TBP–TATA-box complex is +distinctly asymmetric, a property that is crucial for specifying a unique +start site and ensuring that transcription proceeds unidirectionally. + + +TBP bound to the TATA box is the heart of the initiation complex ( **Figure** +**29.25** ). The surface of the TBP saddle provides docking sites for the +binding of other components, with additional transcription factors +assembling on this nucleus in a defined sequence. TFIIA is recruited, +followed by TFIIB; then TFIIF, RNA polymerase II, TFIIE, and TFIIH join +the other factors to form a complex called the _pre-initiation complex_ +(PIC). + + +These additional transcription factors play specific roles in this +complex. As we saw above, TFIID recognizes core promoter elements +and is central to the assembly process. While TFIIA is not essential for +the assembly or function of the PIC in vitro, it may aid in the binding of +TFIID to the DNA. TFIIB is a DNA-binding protein that recognizes +specific cis-acting promoter elements called _B recognition elements_, +which are often found near the TATA box. TFIIF aids in the recruitment +of polymerase II, while TFIIE brings TFIIH to the complex. TFIIH is a +multisubunit complex with helicase and protein kinase activities, both +of which are critical in the initiation of transcription. The helicase +activity unwinds the DNA template, and the kinase activity +phosphorylates specific amino acids in the CTD of polymerase II. + + +During the formation of the PIC, the carboxyl-terminal domain (CTD) is +unphosphorylated and plays a role in transcription regulation through +its binding to an enhancer-associated complex called _mediator_ (Section +31.4). Phosphorylation of the CTD by TFIIH marks the transition from +initiation to elongation. The phosphorylated CTD stabilizes +transcription elongation by RNA polymerase II and recruits RNAprocessing enzymes that act during the course of elongation. The +importance of the carboxyl-terminal domain is highlighted by the +finding that yeast cells containing mutant polymerase II with fewer than +10 repeats in the CTD are not viable. + + +The PIC described above initiates transcription at a low (basal) +frequency, and the transcription factors associated with it are referred + + +to as _basal_ or +_general_ transcription factors. Additional transcription +factors that bind to other sites are required to achieve a high rate of +mRNA synthesis. Their role is to selectively stimulate _specific g_ enes. In +summary, transcription factors and other proteins that bind to +regulatory sites on DNA can be regarded as passwords that +cooperatively open multiple locks, giving RNA polymerase access to + +_._ +specific genes + +###### **Self–Check Question** + + +The function of the σ subunit of _E. coli_ RNA polymerase is analogous to the function of +general transcription factors in eukaryotes. Briefly describe their common function. + +#### **Enhancer sequences can stimulate** **transcription at start sites thousands of** **bases away** + + +The activities of many promoters in higher eukaryotes are greatly +**enhancer** . +increased by another type of cis-acting element called an + + +Enhancer sequences have no promoter activity of their own yet can +exert their stimulatory actions over distances of several thousand +base pairs. + + +Enhancers can be upstream, downstream, or even in the middle of +a transcribed gene _._ + + +Enhancers are effective when present on either the coding or +noncoding DNA strand _._ + + +A particular enhancer is effective only in certain cells; for example, +the immunoglobulin enhancer functions in B lymphocytes but not +elsewhere. + + +Cancer can result if the relation between genes and enhancers is + + +disrupted. In Burkitt lymphoma and B-cell leukemia, a chromosomal +translocation brings the proto-oncogene _myc_ (a transcription factor +itself) under the control of a powerful immunoglobulin enhancer. The +consequent dysregulation of the _myc_ gene is hypothesized to play a role +in the progression of the cancer. + + +The discovery of promoters and enhancers has allowed us to gain a +better understanding of how genes are selectively expressed in +eukaryotic cells. The regulation of eukaryotic gene transcription, +discussed in Chapter 31, is the fundamental means of controlling gene +expression. + + +## **29.4 Some RNA Transcription** **Products Are Processed** + +Virtually all the initial products of eukaryotic transcription are further +processed, and even some prokaryotic transcripts are modified. As we +will see next, the particular processing steps and the factors taking part +vary according to the type of RNA precursor and the type of RNA +polymerase that produced it. + +#### **Precursors of transfer and ribosomal RNA** **are cleaved and chemically modified after** **transcription** + + +In bacteria, messenger RNA molecules undergo little or no modification +after synthesis by RNA polymerase. Indeed, many mRNA molecules are +translated while they are being transcribed. In contrast, transfer RNA +(tRNA) and ribosomal RNA (rRNA) molecules are generated by + +_._ +modifications of nascent RNA chains + + +For +_The transcript can be cleaved at specific sites along its sequence._ +example, in _E. coli_, the three rRNAs and a tRNA are excised from a +single primary RNA transcript that also contains spacer regions +( **Figure 29.26** ). Other transcripts contain arrays of several kinds of +tRNA or several copies of the same tRNA. The nucleases that cleave +and trim these precursors of rRNA and tRNA are highly precise. + + +_Ribonuclease P_ (RNase P), for example, generates the correct + + +terminus of all tRNA molecules in _E. coli._ Sidney Altman and his +coworkers showed that this interesting enzyme contains a +catalytically active RNA molecule. _Ribonuclease III_ (Rnase III) +excises 5S, 16S, and 23S rRNA precursors from the primary + + +transcript by cleaving double-helical hairpin regions at specific +sites. + + + + + +_Nucleotides can be added to the termini of some RNA chains_ . For +example, CCA, a terminal sequence required for the function of all + + +tRNAs, is added to the ends of tRNA molecules for which this + + +terminal sequence is not encoded in the DNA. The enzyme that +catalyzes the addition of CCA is atypical for an RNA polymerase in +that it does not use a DNA template. + + +_Bases and ribose units of RNAs can be modified._ For example, some +bases of rRNA are methylated. Furthermore, all tRNA molecules +contain unusual bases formed by the enzymatic modification of a +standard ribonucleotide in a tRNA precursor. For example, +uridylate residues are modified after transcription to form +ribothymidylate and pseudouridylate. These modifications generate +diversity, allowing greater structural and functional versatility. + + +#### **RNA polymerase I produces three ribosomal** **RNAs** + +Several RNA molecules are key components of ribosomes. In +eukaryotes, RNA polymerase I transcription produces a single precursor +(45S in mammals) that encodes three RNA components of the ribosome: +the 18S rRNA, the 28S rRNA, and the 5.8S rRNA ( **Figure 29.27** ). + + + + + +The 18S rRNA is the RNA component of the small ribosomal subunit +(40S), and the 28S and 5.8S rRNAs are two RNA components of the large +ribosomal subunit (60S). The other RNA component of the large + + +ribosomal subunit, the 5S rRNA, is transcribed by RNA polymerase III as +a separate transcript. Processing of the precursor proceeds as follows: + + +First, the nucleotides of the pre-rRNA sequences destined for the +ribosome undergo extensive modification, on both ribose and base +components, directed by many **small nucleolar** +**ribonucleoproteins (snoRNPs)**, each of which consists of one +snoRNA and several proteins. + + +The pre-rRNA is then assembled with ribosomal proteins, as guided +by processing factors, to form a large ribonucleoprotein. For +instance, the small-subunit (SSU) processome is required for 18S +rRNA synthesis and can be visualized in electron micrographs as a + + +terminal knob at the ends of the nascent rRNAs ( **Figure 29.28** ). + + +Finally, rRNA cleavage (sometimes coupled with additional +processing steps) releases the mature rRNAs assembled with +ribosomal proteins as ribosomes. Like those of RNA polymerase I +transcription itself, most of these processing steps take place in the +cell’s nucleolus. + +#### **RNA polymerase III produces transfer RNAs** + + +Eukaryotic tRNA transcripts are among the most processed of all RNA + + +polymerase III transcripts. Like those of prokaryotic tRNAs, the + + +leader is cleaved by RNase P, the trailer is removed, and CCA is added + + +by the CCA-adding enzyme ( **Figure 29.29** ). Eukaryotic tRNAs are also +heavily modified on base and ribose moieties; these modifications are +important for function. In contrast with prokaryotic tRNAs, many +eukaryotic pre-tRNAs are also spliced by an endonuclease and a ligase +to remove an intron. + + +#### **The product of RNA polymerase II, the pre-** **mRNA transcript, acquires a cap and a** + + +#### **poly(A) tail** + +Perhaps the most extensively studied transcription product is the +product of RNA polymerase II: most of this RNA will be processed to +mRNA. The immediate product of RNA polymerase II is sometimes + +**-** +referred to as precursor-to-messenger RNA, or **pre** **mRNA** . Most premRNA molecules are spliced to remove the introns, which we will + + +discuss in greater detail below. In addition, both the and the ends + +are modified, and both modifications are retained as the pre-mRNA is +converted into mRNA. + + +As in prokaryotes, eukaryotic transcription usually begins with A or G. + + +However, the triphosphate end of the nascent RNA chain is + +immediately modified: + + +First, a phosphoryl group is released by hydrolysis. + + +The diphosphate end then attacks the α -phosphorus atom of GTP + + +to form a very unusual triphosphate linkage. This distinctive + + +terminus is called a **cap** ( **Figure 29.30** ). + + + The N-7 nitrogen of the terminal guanine is then methylated by _S_ +adenosylmethionine to form cap 0. The adjacent riboses may be +methylated to form cap 1 or cap 2. + + +Caps contribute to the stability of mRNAs by protecting their ends + + +from phosphatases and nucleases. In addition, caps enhance the +translation of mRNA by eukaryotic protein-synthesizing systems. +Transfer RNA and ribosomal RNA molecules, in contrast with +messenger RNAs and with small RNAs that participate in splicing, do +not have caps. + + +As mentioned earlier, pre-mRNA is also modified at the end. Most + + +eukaryotic mRNAs contain a string of adenine nucleotides — a **poly(A)** +**tail** — at that end. This poly(A) tail is added _after_ transcription has +ended, since the DNA template does not encode this sequence. Indeed, +the nucleotide preceding poly(A) is not the last nucleotide to be +transcribed. Some primary transcripts contain hundreds of nucleotides + + +beyond the end of the mature mRNA. + + +How is the end of the pre-mRNA given its final form? Eukaryotic + +primary transcripts are cleaved by a specific endonuclease that +recognizes the sequence AAUAAA ( **Figure 29.31** ). Cleavage does not take + + +place if this sequence or a segment of some 20 nucleotides on its side + + +is deleted. The presence of internal AAUAAA sequences in some mature +mRNAs indicates that AAUAAA is only part of the cleavage signal; its +context also is important. After cleavage of the pre-RNA by the +endonuclease, a _poly(A) polymerase_ adds about 250 adenylate residues to + + +the end of the transcript; ATP is the donor in this reaction. + + + + + +The role of the poly(A) tail is still not firmly established despite much +effort. However, evidence is accumulating that it enhances translation +efficiency and the stability of mRNA. Blocking the synthesis of the +poly(A) tail by exposure to -deoxyadenosine (cordycepin) does not + + +interfere with the synthesis of the primary transcript. Messenger RNA + + +without a poly(A) tail can be transported out of the nucleus. However, an +mRNA molecule without a poly(A) tail is usually much less effective as a +template for protein synthesis than one with a poly(A) tail. Indeed, +some mRNAs are stored in an unadenylated form and receive the +poly(A) tail only when translation is imminent. The half-life of an mRNA +molecule may be determined in part by the rate of degradation of its +poly(A) tail. + +#### **Sequences at the ends of introns specify** **splice sites in mRNA precursors** + + +Most genes in higher eukaryotes are composed of exons and introns +(Section 8.7). The introns must be excised and the exons linked to form +the final mRNA in a process called **RNA splicing** . This splicing must be +exquisitely sensitive; splicing just one nucleotide upstream or +downstream of the intended site would create a one-nucleotide shift, + + +which would alter the reading frame on the side of the splice to give + + +an entirely different amino acid sequence, likely including a premature +stop codon. Thus, the correct splice site must be clearly marked. + + +Does a particular sequence denote the splice site? The sequences of +thousands of intron–exon junctions within RNA transcripts are known. +In eukaryotes from yeast to mammals, these sequences have a common +structural motif: the intron begins with GU and ends with AG. The + + +consensus sequence at the splice in vertebrates is AGGUAAGU, where + + +the GU is invariant ( **Figure 29.32** ). At the end of an intron, the + + +consensus sequence is a stretch of 10 pyrimidines (U or C; termed the +_polypyrimidine tract_ ), followed by any base, then by C, and ending with +the invariant AG. Introns also have an important internal site located + + +between 20 and 50 nucleotides upstream of the splice site; it is called + + +the _branch site_ for reasons that will be evident shortly. In yeast, the +branch-site sequence is nearly always UACUAAC, whereas in mammals +a variety of sequences are found. + + +The and splice sites and the branch site are essential for + + +determining where splicing takes place. Mutations in each of these +three critical regions lead to aberrant splicing. Introns vary in length +from 50 to 10,000 nucleotides, and so the splicing machinery may have + +to find the site several thousand nucleotides away. Specific sequences + + +near the splice sites (in both the introns and the exons) play an +important role in splicing regulation, particularly in designating splice +sites when there are many alternatives. Researchers are currently +attempting to determine the factors that contribute to splice-site +selection for individual mRNAs. Despite our knowledge of splice-site +sequences, predicting pre-mRNAs and their protein products from +genomic DNA sequence information remains a challenge. + + +#### **Splicing consists of two sequential** **transesterification reactions** + +The splicing of nascent mRNA molecules is a complicated process. It +requires the cooperation of several small RNAs and proteins that form a +large complex called a **spliceosome** . However, the chemistry of the +splicing process is simple. Splicing begins with the cleavage of the + + +phosphodiester bond between the upstream exon (exon 1) and the + + +end of the intron ( **Figure 29.33** ). The attacking group in this reaction is + + +the group of an adenylate residue in the branch site. A + + +phosphodiester bond is formed between this A residue and the + +terminal phosphate of the intron in a transesterification reaction. + + +Note that this adenylate residue is also joined to two other nucleotides + + +by normal phosphodiester bonds ( **Figure 29.34** ). Hence, a branch + + +is generated at this site, and a lariat (loop) intermediate is formed. + + +The terminus of exon 1 then attacks the phosphodiester bond + + +between the intron and exon 2. In another transesterification reaction, +exons 1 and 2 become joined, and the intron is released in lariat form. +Splicing is thus accomplished by two transesterification reactions rather +than by hydrolysis followed by ligation. + + +Both transesterification reactions are promoted by the pair of bound +magnesium ions, in reactions reminiscent of those for DNA and RNA + +polymerases. The first reaction generates a free group at the + + + end of exon 1, and the second reaction links this group to the + + +phosphate of exon 2. The number of phosphodiester bonds stays the +same during these steps, which is crucial because it allows the splicing +reaction itself to proceed without an energy source such as ATP or GTP. + +#### **Small nuclear RNAs in spliceosomes catalyze** **the splicing of mRNA precursors** + + +The nucleus contains many types of small RNA molecules with fewer +than 300 nucleotides, referred to as **small nuclear RNAs (snRNAs)** . A +few of them — designated U1, U2, U4, U5, and U6 — are essential for +splicing mRNA precursors. The secondary structures of these RNAs are +highly conserved in organisms ranging from yeast to human beings. + + +snRNA molecules are associated with specific proteins to form +complexes termed **small nuclear ribonucleoproteins (snRNPs)** ; +investigators often speak of them as “snurps” ( **Table 29.3** ). SnRNPs and +their role in RNA splicing were discovered by Joan Steitz and Michael +Lerner in 1980. One major piece of evidence suggesting a role for +snRNPs in splicing was the base complementarity between portions of +the U1 snRNA and the splice sites found in the unprocessed mRNAs. + + +**TABLE 29.3 Roles of small nuclear ribonucleoproteins (snRNPs) in the** +**splicing of mRNA precursors** + + + + + + + + + +U1 165 Binds the splice site + + +U2 185 Binds the branch site + + +U5 116 +Binds the splice site and then the splice site + + +U4 145 Masks the catalytic activity of U6 + + +U6 106 Catalyzes splicing + + +SnRNPs associate with hundreds of other proteins (called _splicing_ +_factors_ ) and the mRNA precursors to form the large (60S) spliceosomes. +The large and dynamic nature of the spliceosome made the +determination of the detailed three-dimensional structure a great +challenge. However, with the maturation of cryo-electron microscopy +(Section 4.5), the structures of spliceosomes from several species in a +number of different stages of their function have been determined +( **Figure 29.35** ). These structures have added to our understanding of the +splicing process ( **Figure 29.36** ). + + +1. Splicing begins with the recognition of the splice site by the U1 + + +snRNP. U1 snRNA contains a highly conserved six-nucleotide +sequence, not covered by protein in the snRNP, that base-pairs to + + +the splice site of the pre-mRNA. This binding initiates + + +spliceosome assembly on the pre-mRNA molecule. + + +2. U2 snRNP then binds the branch site in the intron by base-pairing + +between a highly conserved sequence in U2 snRNA and the premRNA. U2 snRNP binding requires ATP hydrolysis. + + +3. A preassembled U4-U5-U6 tri-snRNP joins this complex of U1, U2, + +and the mRNA precursor to form the spliceosome. This association +also requires ATP hydrolysis. Experiments with a reagent that crosslinks neighboring pyrimidines in base-paired regions revealed that + + +in this assembly U5 interacts with exon sequences in the splice + + +exon. +site and subsequently with the + + +4. Next, U6 disengages from U4 and undergoes an intramolecular + +rearrangement that permits base-pairing with U2 as well as + + +interaction with the end of the intron, displacing U1 and U4 from + + +the spliceosome. U4 serves as an inhibitor that masks U6 until the +specific splice sites are aligned. The catalytic center includes two +bound magnesium ions bound primarily by phosphate groups from +the U6 RNA ( **Figure 29.37** ). + + +5. These rearrangements result in the first transesterification + + +reaction, cleaving the exon and generating the lariat + + +intermediate. + + +6. Further rearrangements of RNA in the spliceosome facilitate the + +second transesterification. In these rearrangements, U5 aligns the + + +free exon with the exon such that the -hydroxyl group of the + + +exon is positioned to make a nucleophilic attack on the splice + + +site to generate the spliced product. U2, U5, and U6 bound to the +excised lariat intron are released, completing the splicing reaction. + + +Many of the steps in the splicing process require ATP hydrolysis. How is +the free energy associated with ATP hydrolysis used to power splicing? +To achieve the well-ordered rearrangements necessary for splicing, +ATP-powered RNA helicases must unwind RNA helices and allow +alternative base-pairing arrangements to form. Thus, two features of the +splicing process are noteworthy. First, RNA molecules play key roles in +directing the alignment of splice sites and in carrying out catalysis. +Second, ATP-powered helicases unwind RNA duplex intermediates that +facilitate catalysis and induce the release of snRNPs from the mRNA. + +#### **Mutations that affect pre-mRNA splicing** **cause disease** + + +Mutations in either the pre-mRNA (cis-acting) or the splicing factors +(trans-acting) can cause defective pre-mRNA splicing that manifests in +disease. In fact, mutations affecting splicing have been estimated to +cause at least 15% of all genetic diseases. We will look at two examples +here. + + +First, we will consider the possible effects of cis-acting mutations on +hemoglobin function. Mutations in the pre-mRNA cause some forms of +thalassemia, a group of hereditary anemias characterized by the +defective synthesis of hemoglobin (Section 3.3). Cis-acting mutations + + +or +that cause aberrant splicing can occur at the splice sites in + + +either of the two introns of the hemoglobin β chain or in its exons. + + +Typically, mutations in the splice site alter that site such that the + +splicing machinery cannot recognize it, forcing the machinery to find + + +another splice site in the intron and introducing the potential for a + + +premature stop codon. The defective mRNA is normally degraded rather +than translated. Alternatively, mutations in the intron itself may create a + + +new +splice site; in this case, either one of the two splice sites may be + + +recognized ( **Figure 29.38** ). Consequently, some normal protein can be +made, and so the disease is less severe. + + + + +Second, we will consider the possible effects of trans-acting mutations +on eyesight. Disease-causing mutations may also appear in splicing +factors. Retinitis pigmentosa is a disease of acquired blindness, first +described in 1857, with an incidence of 1/3500. About 5% of the +autosomal dominant form of retinitis pigmentosa is likely due to +mutations in the hPrp8 protein, a pre-mRNA splicing factor that is a +component of the U4-U5-U6 tri-snRNP. How a mutation in a splicing +factor that is present in all cells causes disease only in the retina is not +clear; nevertheless, retinitis pigmentosa is a good example of how +mutations that disrupt spliceosome function can cause disease. + +#### **Most human pre-mRNAs can be spliced in** **alternative ways to yield different proteins** + + +As a result of **alternative splicing**, different combinations of exons from +the same gene may be spliced into a mature RNA, producing distinct +forms of a protein for specific tissues, developmental stages, or +signaling pathways. What controls which splicing sites are selected? The +selection is determined by the binding of trans-acting splicing factors to +cis-acting sequences in the pre-mRNA. Most alternative splicing leads to +changes in the coding sequence, resulting in proteins with different +functions. + + +Alternative splicing provides a powerful mechanism for generating +protein diversity. It expands the versatility of genomic sequences +through combinatorial control. Consider a gene with five positions at +which splicing can take place. With the assumption that these + + +alternative splicing pathways can be regulated independently, a total of + +different mRNAs can be generated. + + +Sequencing of the human genome has revealed that most pre-mRNAs +are alternatively spliced, leading to a much greater number of proteins +than would be predicted from the number of genes. An example of +alternative splicing leading to the expression of two different proteins, +each in a different tissue, is provided by the gene encoding both +calcitonin and calcitonin-gene-related peptide (CGRP; **Figure 29.39** ). In +the thyroid gland, the inclusion of exon 4 in one splicing pathway +produces calcitonin, a peptide hormone that regulates calcium and +phosphorus metabolism. In neuronal cells, the exclusion of exon 4 in +another splicing pathway produces CGRP, a peptide hormone that acts +as a vasodilator. A single pre-mRNA thus yields two very different +peptide hormones, depending on cell type. + + + + + +In the above example, only two proteins result from alternative splicing; +however, in other cases, many more can be produced. An extreme + + +example is the _Drosophila_ pre-mRNA that encodes DSCAM, a neuronal +protein affecting axon connectivity. Alternative splicing of this premRNA has the potential to produce 38,016 different combinations of +exons, a greater number than the total number of genes in the +_Drosophila_ genome. However, only a fraction of these potential mRNAs +appear to be produced, owing to regulatory mechanisms that are not yet +well understood. + + +Several human diseases that can be attributed to defects in alternative +splicing are listed in **Table 29.4** . Further understanding of alternative +splicing and the mechanisms of splice-site selection will be crucial to +understanding how the proteome represented by the human genome is +expressed. + + +**TABLE 29.4 Selected human disorders attributed to defects in** +**alternative splicing** + + +Acute intermittent porphyria Porphobilinogen deaminase + + +Breast and ovarian cancer _BRCA1_ + + +Cystic fibrosis _CFTR_ + + +Frontotemporal dementia protein + + +Hemophilia A Factor VIII + + + +HGPRT deficiency (Lesch–Nyhan +syndrome) + + + +Hypoxanthine-guanine +phosphoribosyltransferase + + + +Leigh encephalomyelopathy Pyruvate dehydrogenase E1 α + + +Severe combined immunodeficiency Adenosine deaminase + + +Spinal muscle atrophy _SMN1_ or _SMN2_ + +#### **Transcription and mRNA processing are** **coupled** + + +Although we have described the transcription and processing of mRNAs +as separate events in gene expression, experimental evidence suggests +that the two steps are coordinated by the carboxyl-terminal domain of +RNA polymerase II. We have seen that the CTD consists of a unique +repeated seven-amino-acid sequence, YSPTSPS. Either,, or both +may be phosphorylated in the various repeats. The phosphorylation +state of the CTD is controlled by a number of kinases and phosphatases +and leads the CTD to bind many of the proteins having roles in RNA +transcription and processing. The CTD contributes to efficient +transcription by recruiting certain proteins to the pre-mRNA ( **Figure** +**29.40** ). These proteins include: + + +1. Capping enzymes, which methylate the guanine on the pre +mRNA immediately after transcription begins + + +2. Components of the splicing machinery, which initiate the excision + +of each intron as it is synthesized + + +3. An endonuclease that cleaves the transcript at the poly(A) addition + + +site, creating a free group that is the target for + + +adenylation + + +These events take place sequentially, directed by the phosphorylation +state of the CTD. + +#### **Small regulatory RNAs are cleaved from** **larger precursors** + + +Cleavage plays a role in the processing of small single-stranded RNAs +**microRNAs** . +(approximately 20–23 nucleotides) called MicroRNAs play + +. +key roles in gene regulation in eukaryotes, as we shall see in Chapter 31 +They are generated from initial transcripts produced by RNA +polymerase II and, in some cases, RNA polymerase III. These +transcripts fold into hairpin structures that are cleaved by specific +nucleases at various stages ( **Figure 29.41** ). The final single-stranded + + +RNAs are bound by regulatory proteins, where the RNAs help target the +regulation of specific genes. + + + + +#### **RNA editing can lead to specific changes in** **mRNA** + +Remarkably, the amino acid sequence information encoded by some +mRNAs is altered after transcription. This phenomenon is referred to as +**RNA editing**, a posttranscriptional change in the nucleotide sequence of +RNA that is caused by processes other than RNA splicing. RNA editing is +prominent in some systems; next, we will consider three examples. + + +RNA editing is key to the process of lipid transport by apolipoprotein B +(apo B). Apo B plays an important role in the transport of +triacylglycerols and cholesterol by forming an amphipathic spherical +shell around the lipids carried in lipoprotein particles (Section 27.3). +Apo B exists in two forms, a 512-kDa _apo B-100_ and a 240-kDa _apo B-48._ +The larger form, synthesized by the liver, participates in the transport of +lipids synthesized in the cell. The smaller form, synthesized by the + + +small intestine, carries dietary fat in the form of chylomicrons. Apo B-48 +contains the 2152 N-terminal residues of the 4536-residue apo B-100. +This truncated molecule can form lipoprotein particles but cannot bind +to the low-density-lipoprotein receptor on cell surfaces. + + +What is the relationship between these two forms of apo B? +Experiments revealed that a totally unexpected mechanism for +generating diversity is at work: the changing of the nucleotide sequence +of mRNA _after_ its synthesis ( **Figure 29.42** ) _._ A specific cytidine residue of +mRNA is deaminated to uridine, which changes the codon at residue +2153 from CAA (Gln) to UAA (stop). The deaminase that catalyzes this +reaction is present in the small intestine, but not in the liver, and is +expressed only at certain developmental stages. + + +RNA editing also plays a role in the regulation of postsynaptic receptors. +Glutamate opens cation-specific channels in the vertebrate central +nervous system by binding to receptors in postsynaptic membranes. +RNA editing changes a single glutamine codon (CAG) in the mRNA for +the glutamate receptor to the codon for arginine (CGG). The substitution + +of Arg for Gln in the receptor prevents, but not, from flowing + + +through the channel. + + +In trypanosomes (parasitic protozoans), a different kind of RNA editing +markedly changes several mitochondrial mRNAs. Nearly half the +uridine residues in these mRNAs are inserted by RNA editing. A guide +RNA molecule identifies the sequences to be modified, and a poly(U) tail +on the guide RNA donates uridine residues to the mRNAs undergoing +editing. + + +DNA sequences evidently do not always faithfully represent the +sequence of encoded proteins; crucial functional changes to mRNA can +take place. RNA editing is likely much more common than was formerly +thought. The chemical reactivity of nucleotide bases — including the +susceptibility to deamination that necessitates complex DNA-repair +mechanisms — has been harnessed as an engine for generating +molecular diversity at the RNA and, hence, protein levels. + + +## **29.5 The Discovery of Catalytic RNA** **Revealed a Unique Splicing** **Mechanism** + +RNAs form a surprisingly versatile class of molecules. As we have seen, +splicing is catalyzed largely by RNA molecules, with proteins playing a +secondary role. RNA is also a key component of ribonuclease P, which +catalyzes the maturation of tRNA by endonucleolytic cleavage of + + +nucleotides from the end of the precursor molecule. Finally, as we + + +shall see in Chapter 30, the RNA component of ribosomes is the catalyst +that carries out protein synthesis. + +#### **Some RNAs can promote their own splicing** + + +The versatility of RNA first became clear from observations of the +processing of ribosomal RNA in a single-cell eukaryote, a ciliated +protozoan in the genus _Tetrahymena._ In _Tetrahymena_, a 414-nucleotide +intron is removed from a 6.4-kb precursor to yield the mature 26S rRNA +molecule. + + +In an elegant series of studies of this splicing reaction, Thomas Cech +and his coworkers established that, in the absence of protein, the RNA +spliced itself to precisely excise the intron. Indeed, the RNA alone is +catalytic and, under certain conditions, is thus a **ribozyme** . More than +1500 similar introns have since been found in species as widely +dispersed as bacteria and eukaryotes, though not in vertebrates. + +**-** +Collectively, they are referred to as group I **self** **splicing introns** . + + +**THOMAS CECH** Fascinated by the structure of chromosomes, Thomas Cech pursued +this topic as a graduate student and postdoctoral fellow. He then accompanied his +wife, Dr. Carol Cech, to the University of Colorado in Boulder (UCB), where they had +both been offered faculty positions. They had met in chemistry class while they were +undergraduate students at Grinnell College. At UCB, Dr. Cech set out to purify +enzymes involved in the splicing of a particular RNA molecule, but he and his + + +coworkers soon discovered that the RNA molecule spliced itself. This fundamental +discovery of RNA catalysis, which changed our view of both modern biochemistry +and our evolutionary past, was recognized with the Nobel Prize in Chemistry in 1989. +Dr. Cech has remained at UCB, teaching with infectious enthusiasm throughout his +career. He also served as an investigator (and president from 2000 to 2009) of the +Howard Hughes Medical Institute (HHMI), a leading private funder of biomedical +research. + + +The self-splicing reaction in the group I intron requires an added +guanosine nucleotide ( **Figure 29.43** ). Nucleotides were originally +included in the reaction mixture because it was thought that ATP or GTP +might be needed as an energy source. Instead, the nucleotides were +found to be necessary as cofactors. The required cofactor proved to be a +guanosine unit, in the form of guanosine, GMP, GDP, or GTP. G +(denoting any one of these species) serves not as an energy source but +as an attacking group that becomes transiently incorporated into the + + +RNA. G binds to the RNA and then attacks the splice site to form a + + +phosphodiester bond with the end of the intron. This + + +transesterification reaction generates a group at the end of the + + +upstream exon. This group then attacks the splice site in a + +second transesterification reaction that joins the two exons and leads to +the release of the 414-nucleotide intron. + + +**FIGURE 29.43 Some introns are capable of self-splicing.** A ribosomal RNA precursor +representative of the group I introns, from the protozoan _Tetrahymena_, splices itself in the +presence of a guanosine cofactor (G, shown in green). A 414-nucleotide intron (red) is released in +the first splicing reaction. This intron then splices itself twice again to produce a linear RNA that +has lost a total of 19 nucleotides. This L19 RNA is catalytically active. + + +[Information from T. Cech, RNA as an enzyme. Copyright © 1986 by Scientific American, Inc. All rights reserved.] + + +Self-splicing depends on the structural integrity of the RNA precursor. +Much of the group I intron is needed for self-splicing. This molecule, +like many RNAs, has a folded structure formed by many double-helical +stems and loops ( **Figure 29.44** ), with a well-defined pocket for binding +the guanosine. Examination of the three-dimensional structure of a +catalytically active group I intron determined by x-ray crystallography +reveals the coordination of magnesium ions in the active site analogous +to that observed in protein enzymes such as DNA polymerase. + + +Analysis of the base sequence of the rRNA precursor suggested that the +splice sites are aligned with the catalytic residues by base-pairing + + +between the _internal guide sequence_ (IGS) in the intron and the and + +exons ( **Figure 29.45** ). The IGS first brings together the guanosine + + +cofactor and the splice site so that the group of G can make a + + +nucleophilic attack on the phosphorus atom at this splice site. The IGS +then holds the downstream exon in position for attack by the newly + + +formed group of the upstream exon. A phosphodiester bond is + + +formed between the two exons, and the intron is released as a linear +molecule. Like catalysis by protein enzymes, self-catalysis of bond +formation and breakage in this rRNA precursor is highly specific. + + +The finding of enzymatic activity in the self-splicing intron and in the +RNA component of RNase P has opened new areas of inquiry and +changed the way in which we think about molecular evolution. As +mentioned in an earlier chapter, the discovery that RNA can be a +catalyst as well as an information carrier suggests that an RNA world +may have existed early in the evolution of life, before the appearance of +DNA and protein. + + +Messenger RNA precursors in the mitochondria of yeast and fungi also +undergo self-splicing, as do some RNA precursors in the chloroplasts of +unicellular organisms such as _Chlamydomonas_ . Self-splicing reactions +can be classified according to the nature of the unit that attacks the +upstream splice site. Group I self-splicing is mediated by a guanosine +cofactor, as in _Tetrahymena_ . The attacking moiety in group II splicing is + +the group of a specific adenylate of the intron ( **Figure 29.46** ). + + +Group I and group II self-splicing resembles spliceosome-catalyzed +splicing in two respects. First, in the initial step, a ribose hydroxyl group + + +attacks the splice site. The newly formed terminus of the + + +upstream exon then attacks the splice site to form a phosphodiester + + +bond with the downstream exon. Second, both reactions are + + +transesterifications in which the phosphate moieties at each splice site +are retained in the products. The number of phosphodiester bonds stays + +constant. + + +Group II splicing is like the spliceosome-catalyzed splicing of mRNA + + +precursors in several additional ways. First, the attack at the splice + + +site is carried out by a part of the intron itself (the group of + + +adenosine) rather than by an external cofactor (G). Second, the intron is +released in the form of a lariat. Third, in some instances, the group II +intron is transcribed in pieces that assemble through hydrogen bonding +to the catalytic intron, in a manner analogous to the assembly of the +snRNAs in the spliceosome. + + +The similarities in mechanism have led to the suggestion that the +spliceosome-catalyzed splicing of mRNA precursors evolved from RNAcatalyzed self-splicing. Group II splicing may well be an intermediate +between group I splicing and the splicing in the nuclei of higher +eukaryotes. A major step in this transition was the transfer of catalytic +power from the intron itself to other molecules. The formation of +spliceosomes gave genes a new freedom because introns were no longer +constrained to provide the catalytic center for splicing. Another +advantage of external catalysts for splicing is that they can be more +readily regulated. + + +However, it is important to note that similarities do not establish +ancestry. The similarities between group II introns and mRNA splicing +may be a result of convergent evolution. Perhaps there are only a +limited number of ways to carry out efficient, specific intron excision. +The determination of whether these similarities stem from ancestry or +from chemistry will require expanding our understanding of RNA +biochemistry. + + +###### **Self–Check Question** + +Compare and contrast the three different splicing mechanisms that have been identified. + +#### **RNA enzymes can promote many reactions,** **including RNA polymerization** + + +In this chapter, we have discussed the role of many different RNA +molecules, including those that promote reactions such as splicing. In +Chapter 30, we will discuss the key roles that RNAs play in protein +biosynthesis, and we will see that ribosomal RNAs have a significant +role in the catalysis of peptide bonds. + + +While the ribosomal RNAs have a role in the polymerization of amino +acids into proteins, some RNAs are also capable of catalyzing the +polymerization of other RNA molecules or even themselves. In vitro +experiments have identified the possibility of self-ligating ribozymes: +RNA molecules capable of joining other, short RNAs to their own end. +The ability of RNAs to direct the synthesis of other catalytic molecules +gives us a glimpse into how, in a primordial RNA world, ribozymes may +have accelerated chemical reactions critical for life. + + +### **Chapter 29 Summary** + +**29.1 RNA Molecules Play Different Roles, Primarily in Gene** +**Expression** + + +RNA molecules are integral to many different cellular functions; +for example, protein biosynthesis. +Some RNAs can direct their own modification. + + +**29.2 RNA Polymerases Catalyze Transcription** + + +RNA polymerases synthesize all cellular RNA molecules +according to instructions given by DNA templates. +The direction of RNA synthesis is, as in DNA synthesis. + + +RNA polymerases, unlike DNA polymerases, do not need a +primer. +RNA polymerase in _E. coli_ is a multisubunit enzyme. The +subunit composition of the holoenzyme is + + +. +and that of the core enzyme is + + +Transcription is initiated at promoter sites. +The σ subunit enables the holoenzyme to recognize promoter +sites. The σ subunit usually dissociates from the holoenzyme +after the initiation of the new chain. +Elongation takes place at transcription bubbles that move along +the DNA template at a rate of about 50 nucleotides per second. +The nascent RNA chain contains stop signals that end +transcription. One stop signal is an RNA hairpin, which is +followed by several U residues. A different stop signal is read by +the _rho_ protein, an ATPase. +In _E. coli,_ precursors of transfer RNA and ribosomal RNA are +cleaved and chemically modified after transcription, whereas + + +messenger RNA is used unchanged as a template for protein +synthesis. + + +**29.3 Transcription Is Highly Regulated** + + +Bacteria use alternate σ factors to adjust the transcription levels +of various genes in response to external stimuli. +Some genes are regulated by riboswitches, structures that form +in RNA transcripts and bind specific metabolites. +Eukaryotic DNA is tightly bound to basic proteins called +histones; the combination is called chromatin. DNA wraps +around an octamer of core histones to form a nucleosome. +There are three types of eukaryotic RNA polymerases in the +nucleus, where transcription takes place: RNA polymerase I +makes ribosomal RNA precursors, II makes messenger RNA +precursors, and III makes transfer RNA precursors. +Eukaryotic promoters are composed of several different +elements. The activity of many promoters is greatly increased +by enhancer sequences that have no promoter activity of their + +own. + + +**29.4 Some RNA Transcription Products Are Processed** + + +The ends of mRNA precursors become capped and + + +methylated during transcription. +A +poly(A) tail is added to most mRNA precursors after the + + +nascent chain has been cleaved by an endonuclease. +The splicing of mRNA precursors is carried out by +spliceosomes, which consist of small nuclear +ribonucleoproteins. Splice sites in mRNA precursors are +specified by sequences at ends of introns and by branch sites +near the ends of introns. + + +RNA editing alters the nucleotide sequence of some mRNAs, +such as the one for apolipoprotein B. + + +**29.5 The Discovery of Catalytic RNA Revealed a Unique Splicing** +**Mechanism** + + +Some RNA molecules undergo self-splicing in the absence of +protein. +Spliceosome-catalyzed splicing may have evolved from selfsplicing. +The discovery of catalytic RNA has opened new vistas in our +exploration of early stages of molecular evolution and the +origins of life. + +### **Key Terms** + + +transcription +RNA polymerase +promoter +transcription bubble +sigma ( σ ) subunit + +consensus sequence +rho ( ρ ) protein +riboswitch + +chromatin + +nucleosome + + carboxyl terminal domain (CTD) +TATA box +transcription factor +enhancer +small nucleolar ribonucleoprotein (snoRNP) + + pre mRNA + + +cap + + +poly(A) tail +RNA splicing +spliceosome +small nuclear RNA (snRNA) +small nuclear ribonucleoprotein (snRNP) +alternative splicing +microRNA + +RNA editing +ribozyme + + self splicing introns + +### **Problems** + + +**1.** Why is RNA synthesis not as carefully monitored for errors +as is DNA synthesis? 1, 2 + + +**2.** What are the functions of RNA polymerases? Select all that +apply. 1, 3 + + +a. Polymerization of polypeptides from RNA transcripts +b. Elongation of ribosomal RNA (rRNA) + +c. Initiation of transcription at promoter sites +d. Elongation of messenger RNA (mRNA) transcripts + +e. Initiation of translation from RNA transcripts + + +**3.** The overall structures of RNA polymerase and DNA +polymerase are very different, yet their active sites show +considerable similarities. What do the similarities suggest +about the evolutionary relationship between these two +important enzymes? 1 + + +**4.** The sequence of part of an mRNA transcript is + + +What is the sequence of the DNA coding strand? Of the DNA +template strand? 1 + + +**5.** Sigma protein by itself does not bind to promoter sites. +Predict the effect of a mutation enabling σ to bind to the +region in the absence of other subunits of RNA polymerase. + + +2 + + +**6.** The molecular weight of an amino acid is approximately 110 +Da, and _E. coli_ RNA polymerase has a transcription rate of +approximately 5050 nucleotides per second. What is the +minimum length of time required by _E.coli_ polymerase for the +synthesis of an mRNA encoding a 100-kDa protein? Round your +answer to the nearest whole number. 1 + + +**7.** The autoradiograph below depicts several bacterial genes +undergoing transcription. Identify the DNA. What are the +strands of increasing length? Where is the beginning of +transcription? The end of transcription? What can you +conclude about the number of enzymes participating in RNA +synthesis on a given gene? 2 + + +a. Splicing occurs while the mRNA is attached to the + +nucleosome. + +b. One mRNA can sometimes code for more than one + + +protein by splicing at alternative sites. +c. Splicing occurs while the mRNA is still in the nucleus. +d. In splicing, intron sequences are removed from the + +mRNA in the form of lariats (loops) and are degraded. +e. 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