Johan Dahlin
60f00a0868
All checks were successful
Deploy Quartz site to GitHub Pages / build (push) Successful in 56s
569 lines
14 KiB
Markdown
569 lines
14 KiB
Markdown
---
|
||
tags:
|
||
- biokemi
|
||
- translation
|
||
- slides
|
||
föreläsare: Ana Luis
|
||
---
|
||
|
||
Chapter 8 and Chapter 20
|
||
Ana Luis
|
||
ana.luis@medkem.gu.se
|
||
2025/04/28
|
||
|
||
---
|
||
|
||
Protein Biosynthesis
|
||
|
||
(Translation)
|
||
Chapter 8 and Chapter 20
|
||
Ana Luis
|
||
ana.luis@medkem.gu.se
|
||
2025/04/28
|
||
|
||
----
|
||
|
||
Learning goals
|
||
|
||
• Explain how nucleic acid information is translated into an amino acid sequence and define the role of aminoacyl tRNA synthetases in this process
|
||
• Protein biosynthesis requires the translation of nucleotide sequences into amino acid sequences
|
||
• Describe features of the genetic code
|
||
• tRNA structure and function
|
||
• Aminoacyl-tRNA synthetases establish the genetic code
|
||
|
||
• Define the role of ribosomes in protein synthesis
|
||
• Structure and function of ribosomes
|
||
• Mechanism of protein synthesis (initiation, elongation, translocation and termination)
|
||
|
||
• Describe how certain chemicals can inhibit protein synthesis
|
||
• Antibiotics and toxins inhibit protein synthesis
|
||
|
||
----
|
||
|
||
Why is important to understand the translation mechanisms?
|
||
![[Pasted image 20251125093128.png]]
|
||
### TRANSLATION
|
||
|
||
#### Inhibition of translation of specific mRNAs can lead to diseasess
|
||
- Fragile X mental retardation syndrome:
|
||
- absence of the set of protein isoforms, derived from
|
||
alternative splicing of the Fragile X mental retardation
|
||
gene 1 (FMR1)
|
||
|
||
#### Bacterial toxins can block of protein biosynthesis protein biosynthesis
|
||
- Diphtheria toxin
|
||
|
||
#### Several antibiotics are inhibitors
|
||
- Streptomycin
|
||
- Tetracycline
|
||
- …..
|
||
|
||
⸻
|
||
|
||
Translation = the process of protein biosynthesis
|
||
|
||
Adenine (A)
|
||
Cytosine (C)
|
||
Guanine (G)
|
||
Uracil (U)
|
||
Nucleic acid
|
||
|
||
→
|
||
|
||
Amino acids
|
||
(A) Alanine
|
||
(R) Arginine
|
||
(N) Asparagine
|
||
(D) Aspartic acid
|
||
(C) Cysteine
|
||
(E) Glutamic acid
|
||
(Q) Glutamine
|
||
(G) Glycine
|
||
(H) Histidine
|
||
(I) Isoleucine
|
||
(L) Leucine
|
||
(K) Lysine
|
||
(M) Methionine
|
||
(F) Phenylalanine
|
||
(P) Proline
|
||
(S) Serine
|
||
(T) Threonine
|
||
(W) Tryptophan
|
||
(Y) Tyrosine
|
||
(V) Valine
|
||
|
||
-----
|
||
|
||
Translation = the process of protein biosynthesis
|
||
|
||
The basics of protein biosynthesis are the same across all kingdoms of life:
|
||
• mRNA is decoded in the 5′-to-3′ direction one codon at a time
|
||
• the protein is synthesized in the amino-to-carboxyl direction
|
||
mRNA
|
||
A U G G U G G C U A A G C G G U G A
|
||
Methionine Valine Alanine Lisine Arginine Stop
|
||
5’ 3’
|
||
Amino group
|
||
Carboxyl group
|
||
Amino group
|
||
Carboxyl
|
||
|
||
⸻
|
||
|
||
tRNA
|
||
|
||
a.a.
|
||
Translation = the process of protein biosynthesis
|
||
• anticodon - portion of the tRNA that base pairs with the codon
|
||
C G I
|
||
3’ 5’
|
||
G C C
|
||
5’ 3’
|
||
Codon
|
||
• codon - three coding bases on the mRNA template
|
||
• transfer RNA (tRNA) - function as adaptor molecules between a codon and
|
||
an amino acid (a.a.)
|
||
Anticodon
|
||
mRNA
|
||
The basics of protein biosynthesis are the same across all kingdoms of life:
|
||
The correct protein biosynthesis requires an accurate recognition of codons by anticodons
|
||
|
||
-----
|
||
|
||
General characteristics of Transfer RNA (tRNA) molecules
|
||
|
||
• Each tRNA is a single chain containing between 73 and 93 nucleotides.
|
||
• The secondary structure resembles a cloverleaf
|
||
(~50% of the nucleotides are base-paired)
|
||
Five groups of bases are not base-paired, but participate in
|
||
hydrogen-bonding interactions:
|
||
• 3′ CCA terminal region (acceptor stem)
|
||
• TψC loop
|
||
• “extra arm”
|
||
• anticodon loop
|
||
• DHU loop
|
||
• The three-dimensional structure is L-shaped
|
||
• tRNAs contain 7 to 15 unusual bases
|
||
• methylated or demethylated derivatives
|
||
of A, U, C, and G
|
||
|
||
⸻
|
||
|
||
• At the 3’ end, an activated amino acid is attached to a hydroxyl group of
|
||
adenosine in the CCA region of the acceptor stem
|
||
• the CCA region has the ability to change its conformation during protein synthesis
|
||
• The anticodon loop is near the center of the sequence
|
||
General characteristics of tRNA molecules
|
||
|
||
⸻
|
||
|
||
Genetic code
|
||
|
||
C G I
|
||
3’ 5’
|
||
G C C
|
||
5’ 3’
|
||
Codon
|
||
Anticodon
|
||
mRNA
|
||
• Genetic code: the relation between the sequence of bases in DNA
|
||
and the sequence of amino acids in proteins
|
||
mRNA
|
||
A U G G U G G C U A A G C G G U G A
|
||
Methionine Valine Alanine Lysine Arginine Stop
|
||
5’ 3’
|
||
DNA
|
||
PROTEIN
|
||
|
||
⸻
|
||
|
||
Amino acids are encoded by groups of three bases starting from a fixed point
|
||
|
||
• Features of the Genetic code:
|
||
– three nucleotides (codon) encode an amino acid
|
||
– has directionality
|
||
– nonoverlapping
|
||
– has no punctuation
|
||
– is degenerate (most amino acids are encoded by more
|
||
than one codon)
|
||
mRNA
|
||
A U G G U G G C U A A G C G G U G A
|
||
Methionine Valine Alanine Lysine Arginine Stop
|
||
5’ 3’
|
||
• 61 codons encode specify amino acids (20 in total)
|
||
• 3 codons are stop codons that designate termination
|
||
of translation.
|
||
|
||
⸻
|
||
|
||
’Wobble’ effect in base-pairing
|
||
|
||
’Wobble’ effect: Some tRNA molecules can recognize more than one codon
|
||
tRNA a.a.
|
||
1
|
||
C G I
|
||
3’ 5’
|
||
G C C
|
||
5’ 3’
|
||
Codon
|
||
Anticodon
|
||
mRNA
|
||
tRNA a.a.
|
||
1
|
||
C G I
|
||
3’ 5’
|
||
G C A
|
||
5’ 3’
|
||
Codon
|
||
Anticodon
|
||
mRNA
|
||
|
||
⸻
|
||
|
||
’Wobble’ effect in base-pairing
|
||
|
||
Anticodons base-pair with codons
|
||
’Wobble’ effect: Some tRNA molecules can recognize more than one codon
|
||
Codons that differ in either of their first two bases (from 5’) must be recognized by different tRNAs.
|
||
The first base of the anticodon (5’) determines the degree of wobble
|
||
• The redundancy, or degeneracy, of the genetic code indicates that recognition of
|
||
the third base of a codon is sometimes less discriminating than the other two
|
||
“wobble” = steric freedom in the pairing of the first base of the anticodon with the third base of the codon
|
||
|
||
⸻
|
||
|
||
’Wobble’ effect in base-pairing
|
||
|
||
“wobble hypothesis”: established hypothesis that predicts the binding of anticodons to codons
|
||
TABLE 30.2 Allowed pairings at the third base of the
|
||
codon according to the wobble hypothesis
|
||
Anticodons base-pair with codons
|
||
C
|
||
C
|
||
G
|
||
|
||
⸻
|
||
|
||
’Wobble’ effect in base-pairing
|
||
|
||
“wobble hypothesis”: established hypothesis that predicts the binding of anticodons to codons
|
||
TABLE 30.2 Allowed pairings at the third base of the
|
||
codon according to the wobble hypothesis
|
||
C
|
||
U
|
||
A/
|
||
G
|
||
|
||
⸻
|
||
|
||
’Wobble’ effect in base-pairing
|
||
|
||
“wobble hypothesis”: established hypothesis that predicts the binding of anticodons to codons
|
||
TABLE 30.2 Allowed pairings at the third base of the codon
|
||
Example: If first base of the anticodon is inosine, the
|
||
anticodon can recognize three different codons
|
||
Inosine is formed by the deamination of adenosine
|
||
• has a heterocyclic nitrogen base that can form
|
||
hydrogen bonds with adenine, cytosine and uracil
|
||
The purine base inosine pairs with cytidine, uridine or adenosine
|
||
|
||
⸻
|
||
|
||
Amino acids required for protein biosynthesis must first be attached to specific tRNA molecules
|
||
|
||
• The attachment of a given amino acid to a particular tRNA establishes the genetic code
|
||
Aminoacyl-tRNA synthetases
|
||
Aminoacyl-tRNA synthetases attach specific amino acids to tRNAs
|
||
tRNA
|
||
amino acid
|
||
Aminoacyl-tRNA
|
||
amino
|
||
• acid
|
||
|
||
⸻
|
||
|
||
Ester linkages couple amino acids to tRNA
|
||
|
||
The process of attaching an amino acid to tRNA is called aminosylation
|
||
CCA arm of tRNA
|
||
• Amino acids are bound to the 3’end of the tRNA via an ester bond
|
||
between the carboxyl group on the amino acid and either the 2’ or 3’
|
||
hydroxyl group of the terminal adenosine of the tRNA
|
||
3’
|
||
Aminoacyl-tRNA: Amino acid bound to tRNA
|
||
3’
|
||
|
||
⸻
|
||
|
||
Each aminoacyl-tRNA synthetase is specific for a given amino acid
|
||
|
||
How aminoacyl-tRNA synthetases evolve to differentiate between different amino acids?
|
||
A closer look at the amino acid threonine, valine and serine
|
||
|
||
⸻
|
||
|
||
Threonyl-tRNA Synthetase contains an activation site
|
||
|
||
Each aminoacyl-tRNA synthetase is specific for a given amino acid
|
||
How aminoacyl-tRNA synthetases evolve to differentiate Threonine, Valine and Serine?
|
||
Activation site
|
||
responsible for activating threonine by binding it
|
||
to adenosine triphospate (ATP) and further
|
||
transfer of these amino acid to the tRNA molecule
|
||
• Aminoacyl-tRNA synthetases have highly
|
||
discriminating amino acid activation sites
|
||
|
||
⸻
|
||
|
||
Each aminoacyl-tRNA synthetase is specific for a given amino acid
|
||
|
||
• To avoid coupling to the incorrect amino acid, threonyl-tRNA synthetase (tRNAThr)
|
||
contains a zinc ion at the active site that binds to the amino and hydroxyl groups
|
||
of threonine
|
||
• Valine is similar in overall structure to threonine but
|
||
lacks the hydroxyl group, so it does not bind to tRNAThr
|
||
• Serine is occasionally linked to tRNAThr because
|
||
of the presence of the hydroxyl group
|
||
(Thr)
|
||
(Val)
|
||
(Ser)
|
||
How aminoacyl-tRNA synthetases evolve to differentiate Threonine, Valine and Serine?
|
||
|
||
⸻
|
||
|
||
Threonyl-tRNA Synthetase contains an activation site and an editing site
|
||
|
||
Activation site
|
||
responsible for activating threonine by binding it
|
||
to adenosine triphospate (ATP) and further
|
||
transfer of these amino acid to the tRNA molecule
|
||
Editing site:
|
||
acts as a profreader and removes any incorrect
|
||
bound amino acid from the tRNA molecule
|
||
|
||
⸻
|
||
|
||
Proofreading by Aminoacyl-tRNA Synthetases Increases the Fidelity of Protein Biosynthesis
|
||
|
||
• Threonyl-tRNA synthetase has an editing site that hydrolyzes Serine if this
|
||
is linked to threonine-tRNA
|
||
• Because Thr contains an extra methyl group, it is sterically excluded
|
||
from the editing site
|
||
• The aminoacylated CCA arm of the tRNA is flexible and can swing out
|
||
of the activation site and into the editing site to remove Ser
|
||
• Most aminoacyl-tRNA synthetases contain editing sites and
|
||
activation sites to ensure very high fidelity.
|
||
Threonyl-tRNA synthetase Thr-tRNA
|
||
|
||
⸻
|
||
|
||
Aminoacyl-tRNA synthetases interaction with tRNA
|
||
|
||
Threonine-tRNA synthetase
|
||
tRNA
|
||
• Threonine-tRNA synthetases bind to both the acceptor stem
|
||
and the anticodon loop of the tRNA
|
||
Aminoacyl-tRNA synthetases assign a particular amino acid to a specific tRNA - the true translators of the genetic
|
||
code
|
||
• Some synthetases recognize their tRNA partners primarily on
|
||
the basis of their anticodons
|
||
• Synthetases may also recognize other
|
||
aspects of tRNA structure that vary among
|
||
different tRNAs
|
||
• many of the recognition sites are loops rich in
|
||
unusual bases
|
||
Number of interactions between tRNA
|
||
and aminoacyl-tRNA synsthetases
|
||
|
||
⸻
|
||
|
||
The ribosome is the site of protein synthesis
|
||
|
||
Ribosomes coordinate the interplay of aminoacyl-tRNAs, mRNA, and proteins
|
||
aminoacyl-tRNAs
|
||
|
||
⸻
|
||
|
||
The E. coli ribosome has a sedimentation coefficient of 70S
|
||
|
||
composed of:
|
||
• large (50S) subunit
|
||
• small (30S) subunit
|
||
34 proteins (L1–L34)
|
||
23S rRNA
|
||
5S rRNA
|
||
21 proteins (S1–S21)
|
||
16S rRNA
|
||
• Two-thirds of the mass of ribosomes is RNA
|
||
• Ribosomal RNA (rRNA) is the catalyst for protein synthesis
|
||
|
||
⸻
|
||
|
||
The ribosome has three binding sites for transfer RNAs
|
||
|
||
• Three tRNA-binding sites:
|
||
• A site (aminoacyl)
|
||
• P site (peptidyl)
|
||
• E site (exit)
|
||
• mRNA fragment is bound within the 30S subunit
|
||
• Each tRNA contacts both 30S and 50S subunits
|
||
|
||
⸻
|
||
|
||
Overview of mechanism of protein biosynthesis
|
||
|
||
Initiation, Elongation, Translocation and Termination
|
||
Initiation
|
||
|
||
⸻
|
||
|
||
Initiation of translation
|
||
|
||
• Each initiator region usually displays:
|
||
• start codon: AUG (Met) or sometimes GUG, rarely UUG
|
||
• purine-rich sequence ~10 nt upstream
|
||
• Bacterial mRNAs often encode several polypeptides
|
||
• Shine–Dalgarno sequence = purine-rich region binding rRNA to position initiator codon in P site
|
||
|
||
⸻
|
||
|
||
Bacterial protein synthesis is initiated by N-formylmethionyl-transfer RNA
|
||
|
||
Steps:
|
||
1. Met linked to tRNAfMet by aminoacyl-tRNA synthetase
|
||
2. Met amino group is formylated
|
||
3. fMet-tRNAfMet placed in P site
|
||
(note: tRNAMet inserts internal Met)
|
||
|
||
⸻
|
||
|
||
Initiation factors (IF1, IF2, IF3)
|
||
1. IF1 + IF3 bind 30S to prevent premature binding to 50S
|
||
2. IF2(GTP) + fMet-tRNAfMet + mRNA bind to 30S → 30S initiation complex
|
||
3. Structural changes release IF1 + IF3
|
||
4. IF2 stimulates 50S binding + GTP hydrolysis → 70S initiation complex
|
||
Streptomycin binds 30S and blocks fMet-tRNAfMet binding (bacteria)
|
||
|
||
⸻
|
||
|
||
Elongation
|
||
|
||
Elongation factors deliver aminoacyl-tRNAs to the ribosome
|
||
|
||
⸻
|
||
|
||
Elongation factors deliver aminoacyl-tRNAs
|
||
|
||
Steps:
|
||
1. EF-Tu-GTP binds aminoacyl-tRNA → delivers to A site
|
||
2. Correct codon recognition → GTP hydrolysis → EF-Tu-GDP leaves
|
||
3. EF-Ts binds EF-Tu-GDP
|
||
4. EF-Ts releases GDP → GTP binds → EF-Tu-GTP regenerated
|
||
Tetracycline binds 30S → blocks aminoacyl-tRNA binding
|
||
|
||
⸻
|
||
|
||
Elongation – peptidyl transferase
|
||
|
||
• Amino group of A-site tRNA attacks carbonyl of P-site peptidyl-tRNA
|
||
• Peptidyl transferase center on 50S catalyzes peptide bond formation
|
||
• Reaction is thermodynamically spontaneous but accelerated by ribosome positioning/orientation
|
||
|
||
⸻
|
||
|
||
Translocation
|
||
|
||
Translocation repositions tRNAs and mRNA
|
||
EF-G (translocase) catalyzes 1-codon movement (requires GTP)
|
||
1. EF-G-GTP binds near A site
|
||
2. GTP hydrolysis → conformational change → peptidyl-tRNA shifts A→P
|
||
After peptide bond formation: chain is in P site (50S), anticodon still in A site (30S)
|
||
|
||
⸻
|
||
|
||
Termination
|
||
|
||
Release factors (RFs):
|
||
• RF1 + RF2 recognize stop codons (UAA, UGA, UAG)
|
||
• RF3 (GTPase) removes RF1/RF2
|
||
RF1/RF2 bind stop codon in A site
|
||
RFs contain conserved GGQ motif with methylated Gln → promotes hydrolysis of ester linkage → releases polypeptide
|
||
|
||
⸻
|
||
|
||
Bacteria: transcription and translation are coupled
|
||
|
||
Minimal time gap between transcription and translation
|
||
Polysome: multiple ribosomes translating one mRNA simultaneously
|
||
|
||
⸻
|
||
|
||
Difference between bacterial and eukaryotic protein biosynthesis
|
||
|
||
Bacteria 70S: 50S+30S
|
||
Eukaryotes 80S: 60S+40S
|
||
Initiation differences:
|
||
• Initiating amino acid = Met (not fMet)
|
||
• No Shine–Dalgarno
|
||
• Start site usually first AUG from 5’ end
|
||
• Many more eIFs
|
||
Eukaryotic mRNA circularization: 5’-cap proteins + PABP at 3’-poly(A) interact → circular mRNA
|
||
|
||
⸻
|
||
|
||
TABLE 30.4 Antibiotic inhibitors of protein biosynthesis
|
||
|
||
Streptomycin/aminoglycosides — inhibit initiation + misreading (bacteria)
|
||
Tetracycline — blocks aminoacyl-tRNA binding (30S)
|
||
Chloramphenicol — inhibits peptidyl transferase (50S)
|
||
Cycloheximide — inhibits translocation (eukaryotes)
|
||
Erythromycin — blocks translocation (50S)
|
||
Puromycin — premature termination (aa-tRNA analog)
|
||
|
||
⸻
|
||
|
||
Diphtheria toxin
|
||
|
||
Blocks protein biosynthesis in eukaryotes by inhibiting translocation
|
||
|
||
⸻
|
||
|
||
Concepts
|
||
|
||
DNA
|
||
RNA
|
||
Protein
|
||
Translation
|
||
Transcription
|
||
Messenger RNA (mRNA)
|
||
Initiation factors
|
||
Elongation factors
|
||
Release factors
|
||
Aminoacyl tRNA synthetases
|
||
Transfer RNA (tRNA)
|
||
Ribosome (ribosomal RNA + proteins)
|
||
EF1 EF2 EF3
|
||
EF-Tu EF-Ts EF-G
|
||
RF1 RF2 RF3
|
||
• Genetic code
|
||
• Degeneracy
|
||
• Codon
|
||
• Anticodon
|
||
• Transfer RNA
|
||
• ’Wobble’ effect
|
||
• Aminoacyl-tRNA synthetases
|
||
• Aminoacyl-tRNA
|
||
• Aminosylation
|
||
• Aminoacyl-tRNA synthetases activation site
|
||
• Aminoacyl-tRNA synthetases editing site
|
||
• Ribosome
|
||
• Ribosomal RNA
|
||
• Ribosome E site, P site and A site
|
||
• Shine-Dalgarno sequence
|
||
• Initiation, elongation, translocation, termination
|
||
• Initiation factors
|
||
• Elongation factors
|
||
• Termination factors
|
||
• Polysome
|
||
• Inhibition of trancription
|