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