#18 Ch24 Translation

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Chapter 24

Translation


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Structure is evolutionarily conserved, yet overall size and

components vary

24.1 Introduction

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Ribosomes are ribonucleoprotein particles.

24.1 Introduction

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An aminoacyl-tRNA brings an amino acidto ribosome and enters the A site.

Peptidyl-tRNA is bound in the P site. Deacylated tRNA exits via the E site(in

bacteria eukaryotic ribosome does nothave E site).

An amino acid is added to the polypeptidechain by transferring the polypeptide frompeptidyl-tRNA in the P site to aminoacyl-tRNA in the A site.

A & P sites occupy both large and smallsubunits of ribosome.

24.2. Translation Occurs byInitiation, Elongation, and

Termination

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Initiation: all the processesbefore forming the peptide bond

between the first two amino

acids of the protein (e.g.,

ribosome binding to mRNA)!

slow and rate-limiting step Elongation: fastest step intranslation. An amino acid isadded to the polypeptide chain

by transferring the polypeptide

from peptidyl-tRNA in the P siteto aminoacyl-tRNA in the A site

(translocation)

Termination: release ofpolypeptide and ribosome

dissociation

24.2. Translation Occurs by Initiation,Elongation, and Termination

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Ribosome and 30S/50Ssubunits are inequilibrium.

30S subunit binds tomRNA first and 50S isrecruited.

Ribosome assembly onmRNA requires initiationfactors (IFs)

Ribosome is releasedas free ribosome orribosomal subunits.

24.4. Initiation in Bacteria Needs 30S

Subunits and Accessory Factors

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Ribosome-binding site (rbs):a short sequence of basesthat precedes the startcodon.

Shine-Dalgarno sequence(AGGAGG): ~10 baseupstream of AUG,

complementary to 3 end of16S rRNA.

Also read section 24.6.

24.4. Initiation in Bacteria

Needs 30S Subunits and

Accessory Factors

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24.4.Initiation in Bacteria

Needs 30S Subunits and

Accessory Factors (=

Initiation Factors, IFs) Initiation factors (IF-1, -2,

and -3), which bind to 30Ssubunits, are required fortranslation initiation.

A 30S subunit carryinginitiation factors binds to aninitiation site on mRNA toform an initiation complex.

IF-2 binds to initiator tRNAand brings it to P siteelement in 30S subunit/mRNA complex

IF-1 binds to near A site andprevents aminoacyl-tRNA

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IF-3 controlsequilibrium between

ribosomal states

IF-3 enables 30S tobind to mRNA

IF-3 checks theaccuracy of recognition

of the first aminoacyl-tRNA

Must be released toallow 50S subunits to

join the 30S/mRNA.

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24.4.Initiation in

Bacteria Needs 30S

Subunits and

Accessory Factors


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Translation starts with a methionineamino acid usually coded by AUG(GUG and UUG can be used in

bacteria).

In bacteria, mitochondria, andchloroplast different methionine

tRNAs are involved in initiation (N-formyl-methionyl-tRNA;fMet-tRNAf)and elongation (Met-tRNAm).

The initiator tRNA has uniquestructural features:

No base pairing of the first base at 5end

Three G-C pairs in the stem precedingthe anticodon loop

Formylated amino acid

24.5 A Special Initiator tRNA Starts the Polypeptide Chain

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Eukaryotic 40S ribosomalsubunits bind to the 5!cap ofmRNA and scan the mRNA untilthey reach the initiation site.

A eukaryotic initiation siteconsists of a ten-nucleotidesequence that includes an AUGcodon (Kozak consensussequence).

Internal Ribosome Entry Site(IRES): ribosome binds directlyto mRNA structure containingAUG start codon

24.7 Small Subunits Scan

for Initiation Sites on

Eukaryotic mRNA

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Initiation factors are required for allstages of initiation, including:

binding the initiator tRNAi (eIF2) 40S subunit attachment to

mRNA

movement along the mRNAjoining of the 60S subunit

eIF2 and eIF3 bind the initiatorMet-tRNAi and GTP.

The complex binds to the 40Ssubunit before it associateswith mRNA.

eIF4G links 5 and 3 end ofmRNA.

24.7 Small Subunits Scan

for Initiation Sites on

Eukaryotic mRNA

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24.8. Elongation Factor Tu (EF-Tu) Loads Aminoacyl-

tRNA into the A Site

EF-Tu is a monomeric G proteinwhose active form (bound to

GTP) binds aminoacyl-tRNA.

The EF-Tu-GTP-aminoacyl-tRNAcomplex binds to the ribosome A

site (P site is occupied). Once codon-anticodon base-

pairing is complete, Tu

hydrolyzes GTP (releasing Tu-

GDP) and tRNA occupies A site

in 50S; Tu-GDP is converted toTu-GTP by EF-Ts.

In eukaryotes, eEF1"& eEF1#$are counterparts of EF-Tu & EF-

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The 50S subunit has peptidyltransferase activity.

The nascent polypeptide chain istransferredfrom peptidyl-tRNAin the P site to aminoacyl-

tRNA in the A site.

Peptide bond synthesisgenerates deacylated tRNA inthe P site and peptidyl-tRNA in

the A site.

24.9 The Polypeptide Chain Is Transferred to

Aminoacyl-tRNA

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24.10 Translocation Moves

the Ribosome

Ribosomal translocationmoves the mRNA throughthe ribosome by three bases.

Translocation movesdeacylated tRNA into the E

site and peptidyl-tRNA intothe P site, and empties the Asite.

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24.10 Translocation Moves the

Ribosome

The hybrid state modelproposes that translocation

occurs in two stages, in which

the 50S moves relative to the30S and then the 30S moves

along mRNA to restore theoriginal conformation.

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24.11 Elongation Factors Bind

Alternately to the Ribosome

Translocation requires EF-G(eEF2 in eukaryotes), whosestructure resembles the

aminoacyl-tRNA-EF-Tu-GTP

complex.

Binding of EF-Tu and EF-G to theribosome is mutually exclusive.

Translocation requires GTPhydrolysis, which triggers a

change in EF-G, which in turn

triggers a change in ribosome

structure.

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24.11 Elongation Factors Bind Alternately to the

Ribosome

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24.12 Three Codons Terminate Translation and Are

Recognized by Protein Factors

Three stop codonsUAG (amber), UAA (ochre) andUGA (opal) terminate translation.

In bacteria they are used most often with relativefrequencies UAA>UGA>UAG.

Termination codons are recognized by release factors(RFs), not by aminoacyl-tRNAs.

RF1, RF2 and RF3 in bacteria; eRF1 and eRF3 ineukaryotes.


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24.12 Three Codons

Terminate Translation and Are


The structures of the class 1release factors (RF1and RF2in

E. coli ) resemble aminoacyl-

tRNA%EF-Tu and EF-G.

RF1 recognizes UAA and UAG;RF2 recognizes UGA and UAA.

These factors bind to A site onlywhen P site is occupied.

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24.12 Three Codons

Terminate Translation and Are


Termination reaction releasesthe polypeptide (protein) but

leaves a deacylated tRNA and

mRNA still associated with

ribosome.

Ribosome recycling factor (RRF)along with EF-G dissociate

ribosome and uncharged tRNA.

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24.12 Three Codons Terminate Translation and Are


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16S rRNA plays an active role in the functions of the 30Ssubunit. It interacts directly with:

mRNA (i.e., rbs)

23S rRNA in the 50S subunit (ribosome assembly)

the anticodon regions of tRNAs in P site and A site 23S rRNA interacts with the CCA terminus of peptidyl-

tRNA in both P site and A site.

Peptidyl transferase activity resides exclusively in the 23SrRNA but not proteins.

24.15 Two rRNAs Play Active Roles in Translation


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24.16 Translation Can Be Regulated

Translation can be regulated by the 5!UTR of the mRNA. Translation may be regulated by the abundance of various

tRNAs =codon usage.

A repressor protein can regulate translation by preventinga ribosome from binding to an initiation codon.

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#18 Ch24 Translation

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