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Peptide Elongation
Aminoacyl-tRNAA-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:
1. Mutation in 23S rRNA, but not in any of the r-proteins,
confer resistance to antibiotics that inhibit peptide bond
formation.
2. Extraction of almost all the protein content of 50S subunit
leaving <5% of r-proteins retains peptidyl transferase
activity. However, treatments that damage RNA abolish
the catalytic activity.
3. 23S rRNA prepared by in vitro transcription can catalyze
the formation of a peptide bond, although with low
efficiency.
Puromycin terminates protein synthesis by acting as an
analogue of a tRNA charged with an aromatic amino acid.
Inhibition of translation by puromycin
Acid-insoluble
NH2
Puromycin
Puro
Peptidyl transferase
Acidsoluble
NH
NH
Puro
Puro
Location of aa-tRNA and
fMet-tRNAf can be
determined by puromycin
release assay
fMet-tRNAf and Met-tRNAf can
be released by puromycin
aa-tRNA, like Met-tRNAm, can
not be released by puromycin
Translocation
This 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.
Models for
translocation
Peptide bond synthesis
Path of tRNA
Active centers in ribosome
Peptidyl
transferase
EF-Tu/Gbinding site
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
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.
The sites interacted with these antibiotics have been
demonstrated 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 rRNA protected by the antibiotics.
Toxins that block protein synthesis:
a-sarcin: an RNAase from Aspergillus that
cleaves at a loop of eukaryotic large rRNA
(corresponding the 23S rRNA of E. coli.)
Ricin (produced by castor seeds): removes a
base 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.
Comparison of EF-Tu and EF-G complexes
Both EF-Tu and EFG are ribosomedependent 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
Aminoacyl-tRNA-EF-Tu-GDPNP
EF-G-GDP
Molecular
mimicry
The ribosomedependent GTPases,
EF-Tu, EF-G, and
RF3, are all
structurally similar.
Their binding sites in
the ribosome may
overlap, and this
ensures that their
binding with the 50S
subunit is in an
orderly manner.
Accuracy of elongation is achieved by:
1. Removing ternary complexes (aa-tRNA-EF-Tu)
bearing the wrong aa-tRNA before GTP hydrolysis;
2. Eliminating the incorrect aa-tRNA before the wrong
aa can be incorporated into the growing polypeptide
chain (proofreading).
Both screens rely on the weakness of incorrect codonanticodon base pairing to ensure dissociation will occur
more rapidly before either GTP hydrolysis or peptide
bond formation.
Accuracy of translation is achieved by:
1. Charging a tRNA only with the correct aa (a function of
aminoacyl-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
Synonym codons:
codons with same
meanings.
The genetic code
is universal, but
exceptions exist.
The set of tRNA
responding to the
various codons for
each amino acid
(codon usage) is
distinctive for each
organism.
Ochre
Amber
Opal
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
code can be accommodated by
isoaccepting species of tRNA that
bind the same amino acid, or by
wobble base pairing (non-WatsonCrick) between 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-methoxyuridine 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
anticodon of Ile-tRNA, which recognizes AUA, AUU, and
AUC, but not AUG, is I.
The surrounding structure of anticodon also influences
recognition of codons, because a change in a base in
some other region of tRNA alters the ability of anticodon
to recognize codons.
Termination of protein synthesis
Termination (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 suppressor mutation
Nonsense mutation
Nonsense suppressor tRNAs
1. 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
In E. coli, amber suppressors
tend to be relatively efficient (1050%), but ochre suppressor are
difficult to isolate and always
much less efficient (< 10%). This
difference may be because the
ochre codon is used most
frequently and suppression of
this codon may be damaging to
E. coli.
Strong missense suppressor is
not favored due to the damaging
effects caused by a general
substitution of aa.
The effectiveness of a suppressor
tRNA depends on the extent of its
competition with the release
factors or normal tRNA, which in
turn is determined by the affinity
between its anticodon and the
target codon, its concentration in
the cell, and other parameters.
The extent of nonsense
suppression by a given tRNA
varies widely depending on the
context of the codon. The base
on the 3’ side of a codon have a
strong effect.
Suppression of frameshift mutation
Compensating 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
includes RNAase E (an endonuclease that makes the first
cleavage for many mRNAs), polynucleotide phosphorylase
(PNPase, a 3’-5’ exonuclease), and helicase. Secondary
structure within mRNA may provide an obstacle to
exonuclease, and this is unwound by the helicase.
Some RNAs have a poly(A) tail (formed by the poly(A)
polymerase) 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
genome of ciliated protozoa or mycoplasma.
Source
Codon
Usual meaning
New meaning
Fruit fly
UGA
Stop
Tryptophan
Mitochondria
AUA
Isoleucine
Methionine
Protozoa
Nuclei
UAA
UAG
Stop
Glutamine
Mycoplasma
UGA
Stop
Tryptophan