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