medical-notes/content/Biokemi/Cellulära processer/Kromatin/Chapter 29 RNA Functions, Biosynthesis, and Processing.md

# **C h a pt e r 2 9**

##### **RNA Functions, Biosynthesis, and Processing**

##### **Outline**

**29.1** RNA Molecules Play Different Roles, Primarily in Gene
Expression


**29.2** RNA Polymerases Catalyze Transcription
**29.3** Transcription Is Highly Regulated
**29.4** Some RNA Transcription Products Are Processed
**29.5** The Discovery of Catalytic RNA Revealed a Unique Splicing
Mechanism

##### **Learning Goals**


_By the end of this chapter, you should be able to:_


1. 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. Splicing of mRNA does not involve any proteins.