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Peptide Elongation. Aminoacyl-tRNA-A-site biding. P. A. Peptide bond formation. This is catalyzed by a peptidyl transferase activity residing in the 23S rRNA. Evidence suggesting that 23S rRNA has peptidyl transferase activity: - PowerPoint PPT Presentation
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Peptide Elongation
Aminoacyl-tRNA-A-site biding
P A
Peptide bond formationThis is catalyzed by a p
eptidyl transferase activi
ty residing in the 23S rR
NA.
Evidence suggesting that 23S rRNA has peptidyl transferase activity:
1. Mutation in 23S rRNA, but not in any of the r-proteins, co
nfer resistance to antibiotics that inhibit peptide bond for
mation.
2. Extraction of almost all the protein content of 50S subunit
leaving <5% of r-proteins retains peptidyl transferase acti
vity. However, treatments that damage RNA abolish the c
atalytic activity.
3. 23S rRNA prepared by in vitro transcription can catalyze t
he formation of a peptide bond, although with low efficien
cy.
Puromycin terminates protein synthesis by acting as an analogue of a tRNA charged with an aromatic amino acid.
NH2
Puro
NH
Puro
NH
Puro
Peptidyl transferase
Puromycin
Inhibition of translation by puromycin
Acid-insoluble
Acid-soluble
fMet-tRNAf and Met-tRNAf can be released by puromycin
aa-tRNA, like Met-tRNAm, can not be released by puromycin
Location of aa-tRNA and fMet-tRNAf can be determined by puromycin release assay
Models for translocation
TranslocationThis step requires EF-G plus GTP. GTP hydrolysis may cause a change in the structure of EF-G, which in turn forces a change in the ribosome structure. GTP hydrolysis is also needed to release EF-G.
Peptide bond synthesis
Path of tRNA
Active centers in ribosome
EF-Tu/G-binding site
Peptidyl transferase
Change of 16S rRNA conformation during protein synthesis
Triggered by joining with 50S subunit, binding of mRNA, binding of tRNA etc.
Conformational change of ribosome during translocation is achieved mainly by alternative base-paring arrangement of rRNA.
Three steps of translation elongation
The sites interacted with these antibiotics have been dem
onstrated to be located in the 16S rRNA (for streptomycin
and tetracyclines) and 23S rRNA (for chloramphenicol) by
primer extension assay used to define the region in the rR
NA protected by the antibiotics.
Antibiotics that block prokaryotic protein synthesis:Streptomycin: inhibits peptide chain initiation and proofreading; increases misreading of pyrimidines.
Tetracyclines: block aminoacyl-tRNA binding to the A site.
Chloramphenicol: blocks peptidyl transferase. It is effective for bacterial and mitochondrial ribosomes.
Erythromycin: inhibits translocation through the ribosome large subunit.
Toxins that block protein synthesis:-sarcin: an RNAase from Aspergillus that cleav
es at a loop of eukaryotic large rRNA (correspon
ding the 23S rRNA of E. coli.)
Ricin (produced by castor seeds): removes a ba
se from eukaryotic large rRNA.
Sites affected by these toxins are located in the
same loop that is protected by elongation factors,
EF-G and EF-Tu.
Aminoacyl-tRNA-EF-Tu-GDPNP EF-G-GDP
Comparison of EF-Tu and EF-G complexes
Both EF-Tu and EF-G are ribosome-dependent GTPases.
EF-Tu forms a ternary complex with tRNA and GTP, while EF-G forms a binary complex with GTP only.
The structure of lower part of EF-G resembles the shape of tRNA in the ternary complex of EF-Tu.
tRNA
Molecular mimicry
The ribosome-depen
dent GTPases, EF-T
u, EF-G, and RF3, a
re all structurally sim
ilar. Their binding sit
es in the ribosome m
ay overlap, and this
ensures that their bi
nding with the 50S s
ubunit is in an orderl
y manner.
Accuracy of elongation is achieved by:
1. Removing ternary complexes (aa-tRNA-EF-Tu) bea
ring the wrong aa-tRNA before GTP hydrolysis;
2. Eliminating the incorrect aa-tRNA before the wrong
aa can be incorporated into the growing polypeptide c
hain (proofreading).
Both screens rely on the weakness of incorrect codon-a
nticodon base pairing to ensure dissociation will occur
more rapidly before either GTP hydrolysis or peptide bo
nd formation.
Accuracy of translation is achieved by:
1. Charging a tRNA only with the correct aa (a function of ami
noacyl-tRNA synthetase, error rate: < 10-5);
2. Specificity of codon-anticodon recognition: proofreading by
ribosome (error rate before proofreading: 10-1-10-2).
Factors affecting accuracy:
Geometry surrounding the A site affected by S12, S4, and S5.
Velocity of peptide bond formation.
Translation factors may also take part.
Overall accuracy of translation: ~ 5 X 10-4/codon
Error rates at different stages of gene expression
Genetic Code
OpalOchre
Amber
The genetic code
is universal, but
exceptions exist.
Synonym codons:
codons with same
meanings.
The set of tRNA res
ponding to the vari
ous codons for eac
h amino acid (codo
n usage) is distincti
ve for each organis
m.
Number of codons for each a.a. does not closely correlate with its frequency of use in proteins.
E. coli
The code is degenerate at the 3rd base
The degeneracy of the genetic cod
e can be accommodated by isoacc
epting species of tRNA that bind th
e same amino acid, or by wobble
base pairing (non-Watson-Crick) b
etween the codon and anticodon.
Wobble base pairs
Standard pairing
G-U wobble pairing
Base modifications affect pairing patterns
Preferential readings of modified bases for some codons
may occur, e.g., uridine-5-oxyacetic acid and 5-methoxy-
uridine recognize A and G more efficiently than U.
U at the 1st position of the anticodon is usually converted
to a modified form; A at that position is always converted
to I.
I pairs with C, U, and A, but not G. The first base of antic
odon of Ile-tRNA, which recognizes AUA, AUU, and AUC,
but not AUG, is I.
The surrounding structure of anticodon also influences r
ecognition of codons, because a change in a base in so
me other region of tRNA alters the ability of anticodon to
recognize codons.
Termination of protein synthesisTermination (stop) codons
UAA (ochre): most commonly used in bacteria.
UGA (opal): causes more errors (1-3% are misread by Try-tRNA).
UAG (amber)
Release factors catalyze termination
RF-1 recognizes UAA and UAG
RF-2 recognizes UGA and UAA.
RF-3, when binds GTP, helps RF-1 and RF-2 bind to and release from the ribosome.
Cleavage of polypeptide from tRNA
Use H2O instead of aminoacyl-tRNA as the acceptor of polypeptide.
RRF (ribosome recycling factor), acts together with EF-G on 50S subunit to cause dissociation of 50S and 30S subunits.
Nonsense suppressor: stop codon suppression
Wild-type
Nonsense mutation
Nonsense suppressor mutation
Nonsense suppressor tRNAs1. Mutation in the anticodon (in E. coli)
2. Mutation outside the anticodon region
For example, a G to A mutation at position 24 in the D stem of tRNATrp, which results in increased stability of the helix, allowing CCA to pair with UGA in an unusual wobble pairing of C with A, probably by altering the conformation of the anticodon loop.
Missense suppressor tRNA mutation
The mutation can be suppressed by insertion either of the original aa or some other aa.
Effects of suppressor mutations
The effectiveness of a suppressor
tRNA depends on the extent of its
competition with the release facto
rs or normal tRNA, which in turn i
s determined by the affinity betwe
en its anticodon and the target co
don, its concentration in the cell,
and other parameters.
The extent of nonsense suppressi
on by a given tRNA varies widely
depending on the context of the c
odon. The base on the 3’ side of
a codon have a strong effect.
In E. coli, amber suppressors te
nd to be relatively efficient (10-5
0%), but ochre suppressor are di
fficult to isolate and always muc
h less efficient (< 10%). This diff
erence may be because the och
re codon is used most frequently
and suppression of this codon m
ay be damaging to E. coli.
Strong missense suppressor is n
ot favored due to the damaging
effects caused by a general sub
stitution of aa.
Suppression of frameshift mutationCompensating base deletion or insertion;
Suppressor mutations in tRNA
tRNA recognizing a 4-base codon (e.g., change the anticodon of tRNAGly from CCC to CCCC).
tRNA that blocks adjacent base by steric hindrance.
Frameshifting as a normal event in natural translation
Common features:
A “slippery” sequence (aminoacyl-tRNA moves +1 or –1 base.)
Ribosome is delayed at the frameshifting site by some ways to allow the aa-tRNA to rearrange its pairing. They include a scarce aminoacyl-tRNA recognizing the adjacent codon, a termination codon recognized slowly by its release factor, and a special conformation of RNA (“pseudonot”.)
Polysomes
mRNAs are translated by multiple ribosomes in tandem.
Transcription and translation occur simultaneously in the bacteria
Rates of transcription and translation are 40 nt/second and 15 aa/second, respectively.
In one gene, there could be 5 initiations per minute and each mRNA may be translated by 30 ribosome.
Polycistronic mRNA
Translation of polycistronic mRNA
Life cycle of mRNA
Degradation of mRNA
Half life of bacterial mRNA: ~2 min.
mRNA degradation may be catalyzed by a complex that in
cludes RNAase E (an endonuclease that makes the first cl
eavage for many mRNAs), polynucleotide phosphorylase
(PNPase, a 3’-5’ exonuclease), and helicase. Secondary s
tructure within mRNA may provide an obstacle to exonucle
ase, and this is unwound by the helicase.
Some RNAs have a poly(A) tail (formed by the poly(A) poly
merase) that acts as the binding site for the nucleases.
The number of times an mRNA is translated is a function of the affinity of the SD region for ribosome and its stability.
Exceptional codons exist in mitochondria (fruit fly,
mammalian, yeast, plant etc.) and the nuclear ge
nome of ciliated protozoa or mycoplasma.
Source Codon Usual meaning New meaning
Fruit fly UGA Stop Tryptophan
Mitochondria AUA Isoleucine Methionine
Protozoa UAA Stop GlutamineNuclei UAG
Mycoplasma UGA Stop Tryptophan