Traduzione II Schoeftner - Moodle@Units · La iniziazione della traduzione Procarioti The initiator...

Latraduzione

ØtRNAsØAminoacil-tRNA sintetasiØRibosoma

Key-componentsduring translation

Introduction

ØIniziazioneØAllungamentoØTerminazione

Theprocessoftranslation

Ilcodicegenetico

Lainiziazionedellatraduzione

Procarioti

àExactpositioningof30Ssubunitonstartcodon

àLoadingofP-sitewithinitiatortRNA

à Recruitmentof50Ssubunit

Shine-Dalgarno sequence


ProcariotiTheinitiatortRNA/tRNA iniziatore

àFirstaminoacidinprocaryoticproteinsisN-formylmethionine

à theinitiatorRNAisspecificforAUG,GUGstartcodons(tRNAifMet)

àtRNAi isloadedwithMethionin:Met-tRNAifMet

àMethioninetRNA transformylaseaddsformyl group(gruppo formile)tomethinin fMet-tRNAifMet

à fMet-tRNAifMet isincorporatedintotheP-siteofthe30Sribosomalsubunit

(aftertranslationfinished,deformylases removeformyl group(gruppo formile),also1-2AA)

Met-tRNA transformilase


Procarioti

3initiationfactors(IFs)thatmediatetheexactloadingoftheinitiatortRNA totheP-siteofthe30Sribosomalsubunit

Exactpositioningof30Ssubunitonstartcodon,assemblyofinitiationcomplex

àLoadingofIF3totheE-siteblocks30SportionoftheE-siteandpreventsprematurefull-assemblyoftheribosome

àIF1blocks30SportionoftheA-site

àGTPase IF2bindsdoIF1andcontactsfMet-tRNAifMet

àmRNAcontacts30Sminorsubunit,fMet-tRNAifMetpairs

withstartcodon =30Sinitiationcomplex

à70Sinitiationcomplexforms:structuralchange– exitofIF3

à stimulationofGTPase activityofIF2(GTPàGDP),affinityofIF2to30Sreduced,IF2andIF1leave30S

à P-siteloadedwithloadedinitiatortRNA,A-sitereadytobeloaded;E-sitefree.


Eucarioti

àminorsubunitassociatedwithloadedinitiatortRNA andeukaryoticinitiationfactors oftranslation(eIFs)

àRecruitmentofthiscomplextomRNAviathe5’Cap

àminorsubunit/initiator tRNA scansonmRNAuntilFIRST startcondon “AUG”

àMajorsubunitloadedtoforminitiatorcomplex

3stepsineukaryoticinitiationoftranslation

1. Ternarycomplex,TC(complesso ternario)

2. 43Spre-initiationcomplex,43SPIC(complesso di preinizio 43S)

3. 48Spre-initiationcomplex48SPIC(complesso dipreinizio 48S)


Eucarioti1. Ternarycomplex(complesso ternario)

àeIF1,eIF5bindE-site;eIF1AbindsA-site:blockingofaberrantloadingofinitiatortRNA toA-siteandprematureassemblyof80SribosomeàeIF2GTPase bindsintitator tRNA=Met-tRNAi

Met

A.40Sribosomalsubunit



2.43Cpre-initiationcomplex

àeIF2bindstoinitiatortRNA andfitsinitiatortRNA intotheP-siteeIF3islargeandassociatedwithminor

subunit,interactswithothereiFs andsupportsassemblyof43SPIC




2.43Cpreinitiation complex


B.mRNA+eIF4initiationfactors

1. 5’CapboundbyeIF4E2. eIF4GbindeIF4EandmRNA3. eIF4AbindseIF4GandmRNA4. eIF4G-Einteractionismajor

regulatorofinitiation;diversefactorscancompetewithbindingtoeIF4Eàregulatesinitiation

5.eIF4BrecruitedthatactivatesthehelicaseactivityofeIF4A.eIF4Ahelicaseopenssecondarystructuresupstreamofstartcodon



2.43Cpre-initiationcomplex


C.48Spre-initiationcomplexmRNAwitheIF4sinteractswith43Cpre-initiationcomplextoformthe48Spre-initiationcomplex

B.mRNA+eIF4initiationfactors

Interaction between intitation factors andPoly-A tailincreases effciency ofinitiation oftranslation

àInteractionofeIF4Gwithpoly-Abindingproteins

-->QualitycheckofmRNA

àcircleformation

àMoreefficientrecyclingofribosomes afteronecycle

oftranslation

Eucarioti

Identification oftheAUGstartcodonby the48SPIC

48SPIC

80SInitiationcomplex

à eIF4A/Bhelicaseactivitymoves30Ssubunit5’à3’tofindAUG(ATPàADP+Pi)

à InitiatortRNA (Met-tRNAiMet)locksinatFIRSTAUG

Seriesofevents:1. eIF1leaves43SPIC2. StructuralchangeofeIF53. ThismediatesGTPhydrolysisbyeIF2;eIF2nolonger

abletointeractwithinitiatortRNAandleavescomplextogetherwitheIF5

Iniziazione;Eucarioti

àNowinitiatortRNA (Met-tRNAiMet)canbeboundbeeIF5B-GTP;

thisstimulatestheassemblyofthe60Swiththe40Ssubunit

à GTPhydrolysisbyeIF5B,eIF5B-GDPandeiF4Aleavecomplex

à InitiatortRNA (Met-tRNAiMet)locatedinP-site;A-siteandE-site

areaccessible=80SInitiationcomplex

READYFORELONGATION

scanning

-i-tR

NAlocksin

-Re-mod

ulation

of43SPIC

eiFfactor

exchange

Allungamento dellatraduzione- Elongation

1.initiationcomplex

2.Correctaminoacyl-tRNA isloadedintoA-site

3.Peptidyl-transferasereaction

4.Translocationofribosome

5.A-siteagainfree

Controlle

dbyelongation

factors(EFs)

TranslationalelongationisHIGHLYCONSERVEDbetweeneukaryotesandprokaryotes

(slidesarefromprokaryotes unlessotherwiseindicated)

Allungamento dellatraduzione- Elongation

TranslationalelongationisHIGHLYCONSERVEDbetweeneukaryotesandprokaryotes

EF-Tu assembles aminoacyl-tRNA into theA-site

EF-Tu isaGTPbindingproteinthatinteractswiththemajorRibosomesubunitandchargedtRNA tocontroltheloadingofaminoacyl-tRNAtotheA-siteandtoprotectsfroma

prematurepeptidyl-transferasereaction

à EF-Tu-GTPbindsto3’-terminusofaminoacyl-tRNA

à aminoacyl-tRNA - EF-Tu-GTPcomplexentersA-siteofmajorsubunit

àOnlywhentheaminoacyl-tRNA anti-codonpairswiththemRNAcodon (minorribosomesubunit)ANDEF-Tu fitsperfectlyintoabindingsite(EF-Tu bindingsite)ofthemajorsubunitEF-Tu-GTPcomplex,GTPase activityisactivated

àGTP-hydrolysisbyEF-Tu GTPase activityresultsinthereleaseofaminoacyl-tRNA;EF-Tu leavesribosometoberecycled.

NEXTSTEP:PEPTIDYL-TRANSFERASEREACTION

Allungamento;Eucarioti

Mechanisms that ensure correct reading ofmRNA codons

1. 2adenineof16SrRNA stabilizecodon-anticodoninteraction

Correctcodon-anticodon pairing

à2adeninein16SrRNA (minorsubunit)canformhydrogenbondswiththeminorgrooveofthepairedcodon-anticodon region

àStabilisation ofaminoacyl-tRNA – EF-Tu-GTPcomplexoncodon

àEF-Tu-GTPinteractswithitsribosomalbindingsiteandactivatesGTPaseactivity

à Peptidyltransferasereaction

ACCURATEZZAIn-Correctcodon-anticodon pairing

àmissmatch betweencodon andanticodonà2adeninein16SrRNA (minorsubunit)

CANNOTformhydrogenbondswiththeminorgrooveofthepairedcodon-anticodon region

àEF-Tu-GTPCANNOTactivateGTPaseactivity

àaminoacyl-tRNA – EF-Tu-GTPcomplexleavesA-siteàNewattemptofcorrectloadingofaminoacyl-tRNA


NEGATIVESELECTION

Inperfectcodon-anticodonPairing:EF-Tu-GTPCANNOTinteractwithEF-Tu bindingsite

2.InteractionofEF-Tu-GTPwithitsribosomal bindingsite(EF-Tu bindingsite)

Perfectcodon-anticodonPairing:EF-Tu-GTPInteractswithribosomalbindingsite

àDE-stabilisation ofaminoacyl-tRNA – EF-Tu-GTPcomplexoncodon

ACCURATEZZA


3.Accomodation ofamminoacyl-tRNA inA-site

NEGATIVESELECTION

àamminoacyl-tRNA isloadedtoA-sitewithaminoacidpointingoutwards.Thispreventsunwantedpeptidyltransferasereactions

ànext,3’terminusofaminiacyl-tRNAwillbetwistedtopull

àA-siteaminoacidclosetonascentpeptidechain

àcorrectcodon-anticodon pairingresistphysicalforce

àImperfectcorrectcodon-anticodon pairing

àPhysicalforceseperates codon fromanitcodon

àAminoacyl-tRNA leavesribosome

ACCURATEZZA

Thepeptidyl-transferase reaction andtranslocation

P-siteA-site

Majorribosomesubunit

ribosomesareRNA:proteincomplexes

PeptidesynthesisIscatalyzedbyribosomalRNA(23SrRNA,majorsubunit)

MechanisminvolvesInteractionbetweenrRNA andtRNA butisunclear

RibosomalproteinL27supportspeptidyl-transferase reaction.L27reachesclosetoactivecenter;DeletionofthesepartsslowdownSynthesis,butdonotabolishpeptidyl-Transferase activity

Thepeptidyl-transferase reaction andtranslocation

A.“Majorsubunitaction”à peptidyl-transferasereaction

à3’endofnewpeptidyl-tRNA movesfromA-sitetoP-siteinmajorsubunit

àCodon-anticodonpairingmaintained

=hybridstate

àMinorsubunitrotatesandallowsnewinteractiontRNA-rRNA

A.“EF-Gaction”àEF-G-GTPbindstoEF-Gbindingsiteafterpeptidyl-transferasereaction

àGTPhydolysis,structuralchangeEF-G-GDPoccupiesA-site

à“gate”betweenAandPsiteopensandpermitspassageofA-sitePeptidyl-tRNA toP-siteP-sitetRNA shiftedtoE-site

àMinorsubunitshiftsbackintooriginalposition

àNewcodonbecomesavailableinA-site(ribosomemovesforward)

EF-Grepresents aEF-Tu-tRNA molecular mimicry

tRNA

GDP

EF-Tu

tRNA mimicry

“mimetism molecolare”

GDP

EF-G

Recycling ofEF-Tu andEF-G

EF-G EF-Tu

àFor eachpeptidyl-transferasereaction1EF-G-GTPand1EF-Tu-GTParehydrolyzedà RecyclingbyexchangingGDPforGTP

à GTPhydrolyzedtoGDP+Pià GDPhaslowaffinityforEF-Gà newGTPreplacesGDPà EF-G-GTPreadyfornextelongationstep

àEF-TsbindsEF-Tu-GDPàreleaseofGDPànewGTPaddedtoEF-Tuàreadyfornextelongation

ENERGYCONSUMPTIONDURINGTRANSLATION

Amminoacyl-tRNA synthetase:1ATPforaddingaminoacidtotRNA 3’end

EF-Tu:1GTPforfittingcorrectaminoacyl-tRNA intoA-site(proofreadingfunction)

EF-G:1GTPtranslocationofribosome(A-P-Esitepassage)

Peptide+1AA:1ATP,2GTPs

TERMINATIONOFTRANSLATION

STOPCODONS:

UAG:codoneAmbra (scopertoda HarrisBernsteinUAA:codoneOcraUGA:codoneOpale

àNotRNA-aminoacyl loadinginA-siteàPolypeptidechainmustbecleavedfrompeptidyl-aminoacyltRNA locatedinP-site

TERMINATIONISA2STEPPROCESSCONTROLLEDBYRELEASEFACTORS(RFs)

Prokaryotes:Class1RFs:RF1:recognizesUAG

RF2:recognizesUGARF1+RF2recognizeUAA

Eukaryotes: onlyoneclassIfactor:eRF1recognizesUAG,UAA,UGA

Step1:Hydrolysisof

Polypeptidechainatpeptidyl-tRNA

Eukaryotes:oneclassIIRF:eRF3

Prokaryotes:oneclassIIRF:RF3 Step2:Releaseofclass1RFs fromribosome

E-siteP-site

RF1boundtoA-site

A-site

TERMINATIONOFTRANSLATIONBYRFs

Class1RFs containPEPTIDEANTICODONs (anticodon peptidico)

à3aminoacids recognizeSTOPcodonsinmRNAtemplateà exchangingpeptideanticodons betweenRF1(UAG)andRF2(UGA)

exchangesstopcodon specificity!!

RF1andRF2structureissimilar

totRNAsPeptideanticodon

interactswithstopcodoninA-site

(otherproteinmotifssupportinteractionofRF1/RF2withribosome)

RF1andRF2structureissimilar

totRNAsàAnticodonpeptide

àGQQmotif


RF1anRF2containaGGQ(Gycin-Glycin-Glutamin)motifthatislocatedclosetothepeptidyl-transferasecenterofthemajorribosomalsubunit

GGQ(Gycin-Glycin-Glutamin)promoteshydrolysisofthenascentpolypeptidechain

ààGGQ(Gycin-Glycin-Glutamin)motifandthepeptideanticodonmimiccentralfunctionsofatRNA


à hydrolysisofpolypeptidechainstimulatedbyclassIRFs

à RF3-GDPisloadedtotheribosomeonlywhenRF1ispresent

à releaseofpolypeptidestimulatedbyclassIRFs

à ThispermitstheexchangeofGDPforGTPinRF3

à Conformationalchange

àThisallowstobindRF3-GTPtoitsbindingsiteinthemajorsubunit;RF1expulsed

GTPhydolysis;RF3-GDPhaslowaffinityforribosomeanddissociate

TRANSLATIONTERMINATED

à Ribosome+E+PsitetRNAs entersintorecycling

RIBOSOMERECYCLING– Riciclaggio delribosoma

RIBOSOMERECYCLINGFACTOR(RRF)

à RRFmimicstRNAandentersA-site

à RRFrecruitsEF-G-GTPintoA-site

àGTP-hydrolysis,PandEsitetRNAswillberemovedandRFFentersP-site(mimicing thetransferoftRNA fromAtoPsite)

àGTP-hydrolysis,PandEsitetRNAswillberemovedandRFFentersP-site(mimicing thetransferoftRNA fromAtoPsite)

àEF-G-GDPleavesmajorsubunit

àEF-G-GDPleavesmajorsubunitandIF3aimstobindtheMinorribosomesubunit

àDisassemblyofribosome,mRNA

àIF3islocatedagaintoE-site

àNEWRIBOSOMECYCLECANINITIATE

THERIBOZYMEISAPREFERREDTARGETFORANTIBIOTICS

à 40%ofantibioticstargettheribosome,1%oftotalantibioticsusefulformedicineà Frequentlybindcomponentsofthetranslationmachinery

à Translationblocked– bacteriumdies

Example:PUROMYCIN:veryefficientanitbioticsàmimicsanaminoacyl-tRNA intheribosomeAsiteà polypeptidechainwillbetransferredtopuromycinàpolypeptide-puromycin releasedfromribosome

à Antibioticstakeadvantageoftherequirementforpreciseribosomestructurefortranslation(differencesinstructurecanmediateantibioticsresistance!!)

TheuseofribosometargetingantibioticsisastrongtooltoUnderstandtheribosomefunction

à Antibioticsareproducedbybacteriaandfunghi

PUROMYCINtargetspro-andeukaryoteribosomes

THERIBOZYMEISAPREFERREDTARGETFORANTIBIOTICS

Traduzione II Schoeftner - Moodle@Units · La iniziazione della traduzione Procarioti The initiator...

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