TranslationTranslation

Chapter 9Chapter 9

OverviewOverview Occurs on ribosomes-large Occurs on ribosomes-large

aggregates of rRNA and proteinaggregates of rRNA and protein tRNA acts as amino acid carrierstRNA acts as amino acid carriers Prokaryotes—occurs simultaneously Prokaryotes—occurs simultaneously

with transcription and mRNA with transcription and mRNA degradationdegradation

Eukaryotes—occurs in cytoplasmEukaryotes—occurs in cytoplasm mRNA translated 5’mRNA translated 5’3’3’ Protein synthesis aminoProtein synthesis aminocarboxycarboxy

Protein SynthesisProtein Synthesis Polymerization of amino acids: Polymerization of amino acids:

condensation reaction (dehydration condensation reaction (dehydration synthesis)synthesis)

~Universal Genetic Code~Universal Genetic Code Codons—sets of 3 nucleotides Codons—sets of 3 nucleotides

corresponding to a single amino acidcorresponding to a single amino acid Each codon specifies a single amino acidEach codon specifies a single amino acid More than one codon can specify the same More than one codon can specify the same

amino acidamino acid code is said to be code is said to be degeneratedegenerate Some aa correspond to a single codonSome aa correspond to a single codon

AUG—initiator codon, methionine (Met, M)AUG—initiator codon, methionine (Met, M) UGG–Tryoptophan (TrP, W)UGG–Tryoptophan (TrP, W)

Often codons encoding the same aa differ Often codons encoding the same aa differ onl;y at the 3onl;y at the 3rdrd nucleotide nucleotide

~Universal Genetic Code~Universal Genetic Code

GUG sometimes used as a startGUG sometimes used as a start Mammalian mitochondriaMammalian mitochondria Ciliated protozoaCiliated protozoa SelenocysteineSelenocysteine

Why~Universal? ExceptionsWhy~Universal? Exceptions

NH3+

COO-

H-C-CH2Se

SelenocysteineSelenocysteine The 21The 21stst amino acid? amino acid? An essential amino acid for selenoproteins An essential amino acid for selenoproteins

EX. Glutathione oxidaseEX. Glutathione oxidase Uses unique tRNA (tRNASec), initially Uses unique tRNA (tRNASec), initially

bound to Ser. Longest known tRNA (95nt).bound to Ser. Longest known tRNA (95nt). Anticodon recognizes UGA (Stop) as SecAnticodon recognizes UGA (Stop) as Sec Signal (a stem loop configuration) 3’ to Signal (a stem loop configuration) 3’ to

the UGA determine Stop or Secthe UGA determine Stop or Sec Dedicated specific elongation factor Dedicated specific elongation factor

recognizes the stem-loop and substitutes recognizes the stem-loop and substitutes for usual elongation factor (EF-Tu)for usual elongation factor (EF-Tu)

Degeneracy—Wobble Degeneracy—Wobble HypothesisHypothesis

Explains how some tRNA recognize more Explains how some tRNA recognize more than one codonsthan one codons

tRNA molecules only need to make strong tRNA molecules only need to make strong base pairs with 2 of the three codons in base pairs with 2 of the three codons in the nucleotide the nucleotide

This third loose base pairing interaction is This third loose base pairing interaction is called wobblecalled wobble

Note: only certain bases can substitute for Note: only certain bases can substitute for othersothers

Wobble ExampleWobble Example

This UCA codon was read by thetRNA with a UGA anticodonBut if this UCA was

UCG, it would still have been read by the tRNA with a UGA anticodon

Codon Usage Codon Usage More than one codon exists for most amino More than one codon exists for most amino

acids (except Met and Trp)acids (except Met and Trp) Organism may have a preferred codon for a Organism may have a preferred codon for a

particular amino acid particular amino acid Codon usage correlates with abundance of Codon usage correlates with abundance of

tRNAs (preferred codons are represented by tRNAs (preferred codons are represented by abundant tRNAs) abundant tRNAs)

Rare tRNAs correspond to rarely used codonsRare tRNAs correspond to rarely used codons mRNAs containing rare codons experience mRNAs containing rare codons experience

slow translationslow translation Implications for GE?Implications for GE?

Amino Acyl Synthetase and Amino Acyl Synthetase and tRNAtRNA

Amino acyl synthetases catalyze Amino acyl synthetases catalyze attachment of aa to its appropriate attachment of aa to its appropriate tRNAtRNA One for each amino acidOne for each amino acid

tRNAtRNA Derived from large 1Derived from large 1ºº transcript transcript Heavily modified, unusual basesHeavily modified, unusual bases Extensive folding due to internal H-Extensive folding due to internal H-

bonding bonding

Amino Amino Acyl Acyl

SynthetasSynthetasee Carboxy end of aa Carboxy end of aa

attached to attached to --phospate of ATPphospate of ATP

AMP released as AMP released as carboxy end of carboxy end of amoinoacyl group amoinoacyl group transferred to O at C-transferred to O at C-3 of 3’nt3 of 3’nt

When aa is attached, When aa is attached, tRNA is tRNA is chargedcharged or or acylatedacylated

No aa =No aa = uncharged uncharged Wrong aa =Wrong aa =

mischargedmischarged NOTE PPiNOTE PPi

tRNA Activation by Aminoacyl tRNA tRNA Activation by Aminoacyl tRNA SynthetasesSynthetases

C+H3N

R

O

O(-) PO

O

OH OH

O O-

AdenineO

PO

PO(-)

HO

O

O

(-)O

C+H3N

R

O

OP

O

O

OH OH

O O-

Aminoacyl adenylate (Aminoacyl-AMP)

+ PPiAdenine

1. Aminoacyl-AMP formation:

2. Aminoacyl transfer to the appropriate tRNA:

C+H3N

R

O

OAMP+ACC-tRNAC+H3N

R

O

OP

O

O

OH OH

O O-

HO-ACC-tRNAAdenine +

2Pi

Overall reaction: amino acid + tRNA + ATP aminoacyl-tRNA + AMP + PPi

tRNA Function and StructuretRNA Function and Structure Anticodon-Anticodon-

complementary to complementary to codon on mRNAcodon on mRNA

Amino attachment Amino attachment (CCA) site(CCA) site

Other recognition Other recognition sitessites DHU loopDHU loop TTC LoopC Loop Extra arm Extra arm

(variable)(variable) NOTE: also unusual NOTE: also unusual

bases observed bases observed acceptor stemacceptor stem

acceptor stem

tRNA Recognition by Amino tRNA Recognition by Amino Acyl SynthetaseAcyl Synthetase

Sequence elements in each tRNA are Sequence elements in each tRNA are recognized by its specific synthetase recognized by its specific synthetase including:including: One or more bases in acceptor stemOne or more bases in acceptor stem Base at position 73 “Discriminator base”Base at position 73 “Discriminator base”

Seems to play a major role in many cases, Seems to play a major role in many cases, but in other cases it is completely ignored.but in other cases it is completely ignored.

In many, at least one anticodon base In many, at least one anticodon base

Recognition (cont’d)Recognition (cont’d)No common set of rules for tRNA No common set of rules for tRNA

recognition !!!recognition !!! Anticodon region is not the only Anticodon region is not the only

recognition site recognition site The "inside of the L" and other regions The "inside of the L" and other regions

of the tRNA molecule are also important of the tRNA molecule are also important Specificity of several aminoacyl-tRNA Specificity of several aminoacyl-tRNA

synthetases determined by: synthetases determined by: one or more bases in anticodonone or more bases in anticodon one or more bases in the acceptor stemone or more bases in the acceptor stem discriminator base 73discriminator base 73

Mischarging Mischarging Observation: several aa similar in size and shape, Observation: several aa similar in size and shape,

but mischarging rare.but mischarging rare. Editing carried out by aminoacyl tRNA synthetaseEditing carried out by aminoacyl tRNA synthetase Ex Double sieve of isoleucine synthetaseEx Double sieve of isoleucine synthetase

Activation site– coarse sieve, rejects aa larger Activation site– coarse sieve, rejects aa larger than ile. than ile. excluded because they don’t fit.excluded because they don’t fit.

Editing (hydrolytic) site—fine sieve. Accepts Editing (hydrolytic) site—fine sieve. Accepts activated amino acids that are smaller than ile activated amino acids that are smaller than ile (ex, Val-AMP), but rejects Ile-AMP (too large). (ex, Val-AMP), but rejects Ile-AMP (too large). those that get through are hydrolyzed to aa those that get through are hydrolyzed to aa and AMP. Reduces mischarging from 1/225 and AMP. Reduces mischarging from 1/225 (expected) to 1/180,000 (observed). (expected) to 1/180,000 (observed).

Sites can also distinguish based on hydrophobicitySites can also distinguish based on hydrophobicity

Isoleucil-tRNA Synthetase: Isoleucil-tRNA Synthetase: Proofreading Based on SizeProofreading Based on Size

CH3

O+H3N

tRNAIleO

CH3

Smaller Hydrolytic Site

Larger Acylation Site

CH3H3C

ONH3+

O

Larger Acylation Site

Smaller Hydrolytic Site

tRNAIle

CH3

O+H3N

tRNAIleO

CH3

Correct Acylation

H3C CH3

O+H3N

tRNAIleO

Misacylation

Difference in Size

Ile Val

Valyl tRNAValyl tRNAValVal Synthetase Synthetase Proofreading: Proofreading:

Hydrophobic/Polar Recognition MotifHydrophobic/Polar Recognition Motif

3HC CH3

O+H3N

tRNAValO

Hydrophobic Acylation Site

Polar Hydrolytic Site

CH3 CH3

O+H3N

tRNAValO

Correct Acylation

HO CH3

O+H3N

tRNAValO

Misacylation

OHH3C

ONH3+

OtRNAVal

Polar Hydrolytic Site

Hydrophobic Acylation Site

Difference in Hydrophobicity

Val Thr

Experiment (1962)Experiment (1962)

tRNA-ACAAnticodon (recognizes UGU codon, encodes Cys)

Cell-free extract amino acids & enymes

Cys-tRNA-ACA Protein has Cys

RNA template

UGUGUGUGUG...

Treat w metal catalyst removes thiol groups

Ala-tRNA-ACA Protein has Ala

RNA template

UGUGUGUGUG...

tRNA is charged with Cys

Charged amino acid is changed chemically

Once an aminoacyl-tRNA has been synthesized the amino acid part makes no contribution to accurate translation of the mRNA.

Protein Synthesis-3 StagesProtein Synthesis-3 Stages

InitiationInitiation ElongationElongation TerminationTermination

Ribosomes Ribosomes

Composition of Composition of eukaryotic and eukaryotic and prokaryotic prokaryotic ribosomesribosomes

Mol. Biol. Gene, Fig. 14-13

Composition of the E. coli Ribosome

50S subunit 23S & 5S RNA + 34 proteins

30S subunit 16S RNA + 21 proteins

Central protuberance

stalk

ridge

headstalk

platform

platform

Gross anatomy of the E. coli ribosome.

Fig. 19.5

InitiationInitiation

In both prokaryotes and In both prokaryotes and eukaryotes, protein eukaryotes, protein synthesis begins with a synthesis begins with a specific initiating tRNAspecific initiating tRNA

In prokaryotes, initiator In prokaryotes, initiator methionine amino group is methionine amino group is methylatedmethylated Attached to special tRNA Attached to special tRNA

(fMet(fMet--tRNA)tRNA) Transformylase—adds Transformylase—adds

formyl groupformyl group Deformylase—removes Deformylase—removes

formyl group from Met of formyl group from Met of completed peptidecompleted peptide

Formylation does not occur Formylation does not occur in eukaryotesin eukaryotes

Steps in InitiationSteps in Initiation Association of 30S subunit withAssociation of 30S subunit with

mRNAmRNA fMet-tRNAfMet-tRNA Initiation factors (3 proteins)Initiation factors (3 proteins) GTPGTP

QUESTION: AUG encodes fMet and QUESTION: AUG encodes fMet and Met. How does 30S ribosome “know” Met. How does 30S ribosome “know” which aa is to be inserted?which aa is to be inserted?

}30S Pre-initiation Complex

Initiator Codon RecognitionInitiator Codon Recognition fMet-tRNA responds only to initiator fMet-tRNA responds only to initiator

codons (AUG, GUG, UUG [rarely])codons (AUG, GUG, UUG [rarely]) Met-tRNA responds only to internal AUGMet-tRNA responds only to internal AUG Meaning of codons dependent on their Meaning of codons dependent on their

context, i.e., sequences nearbycontext, i.e., sequences nearby In Eukaryotes: 5’ cap involvementIn Eukaryotes: 5’ cap involvement In Prokaryotes: Shine-Dalgarno In Prokaryotes: Shine-Dalgarno

SequenceSequence mRNA- (5’)AGGAG (3’)mRNA- (5’)AGGAG (3’) 16S rRNA- (3’)UCCUC(5’)16S rRNA- (3’)UCCUC(5’)

Shine-Dalgarno Interaction Shine-Dalgarno Interaction Upstream Upstream

from from initiator initiator AUGAUG

ComplemenComplementary to a tary to a stretch on stretch on 16S rRNA16S rRNA

Seen in Seen in virtually all virtually all prokaryotic prokaryotic mRNAmRNA

Initiation Factors in Protein Initiation Factors in Protein SynthesisSynthesis IF-1 IF-1

Promotes dissociation of ribosome. Promotes dissociation of ribosome. IF-1 also blocks the A site of the small IF-1 also blocks the A site of the small

ribosomal subunit ribosomal subunit

insures the initiation aa-tRNA fMet-insures the initiation aa-tRNA fMet-tRNAtRNAfMetfMet can bind only in the P site & that can bind only in the P site & that no other aa-tRNA can bind in the A site no other aa-tRNA can bind in the A site during initiation.during initiation.

IF-2 IF-2 small GTP-binding protein (a GTPase). small GTP-binding protein (a GTPase). Interacts withInteracts with

Small subunitSmall subunit IF1IF1 fMet-tRNAfMet-tRNAfMetfMet

Initiation Factors in Protein Initiation Factors in Protein SynthesisSynthesis

IF-2 (cont’d)IF-2 (cont’d) IF-2/GTP helps the initiator helps it dock with IF-2/GTP helps the initiator helps it dock with

the small ribosome subunit, prevents other the small ribosome subunit, prevents other tRNAs from binding small subunit tRNAs from binding small subunit

IF-3 IF-3 Binds small subunit, prevents reassociation Binds small subunit, prevents reassociation

w/ large subunitw/ large subunit binds mRNA to the 30S ribosomal subunitbinds mRNA to the 30S ribosomal subunit frees it from its complex with the 50S frees it from its complex with the 50S

subunit. subunit.

30S Pre-initiation Complex30S Pre-initiation Complex

70S Initiation Complex 70S Initiation Complex AssemblyAssembly

30S pre-initiation= 30S subunit,IF1-3, mRNA, GTP, 30S pre-initiation= 30S subunit,IF1-3, mRNA, GTP, fMet-tRNAfMet-tRNAfMetfMet

When fMet-tRNAWhen fMet-tRNAfMet fMet pairs with initiator codon, pairs with initiator codon, small subunit undergoes conformational changesmall subunit undergoes conformational change

Result: Release of IF-3Result: Release of IF-3 Large subunit can bind small subunit complexLarge subunit can bind small subunit complex

Binding of large subunit stimulates GTPAse Binding of large subunit stimulates GTPAse activity of IF-2/GTPactivity of IF-2/GTP hydrolysis of GTP to GDPhydrolysis of GTP to GDP

IF-2/GDP and IF-1 fall offIF-2/GDP and IF-1 fall off RESULT: 70S ribosome RESULT: 70S ribosome with fMet-tRNAwith fMet-tRNAfMet fMet in P- in P-

sitesite of ribosome of ribosome

RESULT: RESULT: 70S 70S

Initiation Initiation ComplexComplex

OverviewOverview Dissociation of Dissociation of

inactive 70Sinactive 70S IF-1, IF-3IF-1, IF-3 IF-2/tRNA, mRNAIF-2/tRNA, mRNA fMet-tRNAfMet-tRNAfMet/fMet/initiator initiator

codon-releases IF-3codon-releases IF-3 GTP Hydrolysis, GTP Hydrolysis,

releases IF-1, IF-2releases IF-1, IF-2 Complete 70S complexComplete 70S complex

70S Ribosome70S Ribosome

Large subunit(50 S)

Messenger RNA

Transfer RNAs

P (peptidyl) site

5’ end

A (aminoacyl) site

Small subunit

Alternative ViewAlternative View A=AminoacyA=Aminoacy

l sitel site P=peptidyl P=peptidyl

sitesite E=Exit siteE=Exit site

Elongation Overview Elongation Overview Aminoacyl tRNA complementary to codon Aminoacyl tRNA complementary to codon

in A-site moves into A-sitein A-site moves into A-site N-formyl-Met transferred from tRNAN-formyl-Met transferred from tRNAfMetfMet to to

aminoacyl-tRNA in A-siteaminoacyl-tRNA in A-site now have a dipeptide in the A-sitenow have a dipeptide in the A-site

tRNAtRNAfMetfMet leaves P-site leaves P-site Ribosome moves along mRNA Ribosome moves along mRNA

(translocation)(translocation) dipeptide in P-sitedipeptide in P-site

New aminoacyl-tRNA moves into A site, etcNew aminoacyl-tRNA moves into A site, etc

ElongationELONGATION

O

OHO

HH

H

CH2

H

OPO

O

O

Adenine

tRNA

C

HC

NH

R

O

OHO

HH

H

CH2

H

OPO

O

O

Adenine

tRNA

C

HC

NH2

R

C

HC

NH3+

R

O

O

O

:

P site A site

Transpeptidation Reaction Transpeptidation Reaction

Carboxy end of nascent peptide

Amino terminus of incoming amino acid

Nucleophilic attack

The nascent polypeptide, one residue longer, is now linked to the tRNA in the A site.

O

OHOH

HH

H

CH2

H

OPO

O

O

Adenine

tRNA

O

OHO

HH

H

CH2

H

OPO

O

O

Adenine

tRNA

C

HC

NH

R

O

C

HC

NH

R

C

HC

NH3+

R

O

O

P site A site

Transpeptidation CompletedTranspeptidation Completed

EF-G-EF-G-GTPGTP

Role in Role in translocationtranslocation

Binding site Binding site uncovereduncovered

EF-G-GTP EF-G-GTP occupies occupies

HydrolysisHydrolysis GDP leavesGDP leaves open A-siteopen A-site

Translation Translation TerminationTermination

““Stop” CodonStop” Codon No anticodons, No anticodons,

but are release but are release factors factors ProteinsProteins Occupy A-siteOccupy A-site Activate Activate

hydrolysis of hydrolysis of peptide from peptide from peptidyl-tRNApeptidyl-tRNA

Translation Translation Termination 2Termination 2 Release factorsRelease factors

RF-1 and RF-2 RF-1 and RF-2 recognize stop recognize stop codonscodons

RF-3 –stimulates RF-3 –stimulates dissociation of 70S dissociation of 70S ribosome after ribosome after release of release of polypeptide chainpolypeptide chain

Anticodon Anticodon recognition recognition determined 3 aadetermined 3 aa

Proofreading Proofreading in Translationin Translation

1.1. Codon:anticodon Codon:anticodon base pairingbase pairing

2.2. 16S rRNA forms 16S rRNA forms H-bonds with H-bonds with minor groove of minor groove of codon:anticodon codon:anticodon duplex only when duplex only when correctly pairedcorrectly paired

3.3. Proofreading Proofreading in Translationin Translation

Correct base pairing Correct base pairing allows EF-Tu bound allows EF-Tu bound to aa-tRNA to interact to aa-tRNA to interact with with factor binding factor binding center, center, inducing GTP inducing GTP hydrolysis and EF-Tu hydrolysis and EF-Tu releaserelease

Incorrect base pairingIncorrect base pairing FBC not contactedFBC not contacted allows more time for allows more time for

EF-Tu GTP releaseEF-Tu GTP release

4.4. Proofreading Proofreading in Translationin Translation Incorrectly Incorrectly

paired tRNA paired tRNA can’t rotate can’t rotate into position into position for peptide for peptide bond bond formation formation

““tRNA tRNA accommodatioaccommodation”n”

AntibioticsAntibiotics Translation the target of many Translation the target of many

antibiotics. EXantibiotics. EX

Site of Nucleophilic attack

Absentterminates translation

Eukaryotic Translation Eukaryotic Translation FactorsFactors

designated with the prefix "designated with the prefix "ee" " EF-Ts replaced byEF-Ts replaced by

eEF-1eEF-1 eEF-2 (target for diphtheria toxin)eEF-2 (target for diphtheria toxin)

Diphtheria ToxinDiphtheria Toxin

EF2 is only known substrate for diphtheria EF2 is only known substrate for diphtheria toxintoxin

EF2 contains rare modification of one of histidine EF2 contains rare modification of one of histidine residues and this is site recognized by toxinresidues and this is site recognized by toxin

Mutant cells that cannot modify site are resistantMutant cells that cannot modify site are resistant

Addition of ADP-ribose inactivates EF2Addition of ADP-ribose inactivates EF2 Kills cells by irreversible block of protein synthesisKills cells by irreversible block of protein synthesis

P. aeruginosaP. aeruginosa exotoxin A works same as exotoxin A works same as diphtheria toxindiphtheria toxin