Protein synthesis

Protein synthesis,genetic code with f

1. Page 1
Protein synthesis
Made By-
Dr. NidhiSharma

2. Page 2
•Transcription
•Genetic code
•tRNA
•Ribosomes
•Translation
•Polyribosomes

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Transcription

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•Transcription,orRNAsynthesis,isthe
processofcreatinganequivalentRNAcopy
ofasequenceofDNA.
•BothRNAandDNAarenucleicacids,which
usebasepairsofnucleotidesasa
complementarylanguagethatcanbe
convertedbackandforthfromDNAtoRNA
inthepresenceofthecorrectenzymes.

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Central dogma
DNA RNA
Proteins
Transcription Translation

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•Duringtranscription,aDNAsequenceis
readbyRNApolymerase,whichproducesa
complementary,antiparallelRNAstrand.
•AsopposedtoDNAreplication,
transcriptionresultsinanRNAcompliment
thatincludesuracil(U)inallinstances
wherethymine(T)wouldhaveoccurredin
aDNAcompliment.

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•Transcriptionisthefirststepleadingtogene
expression.
•ThestretchofDNAtranscribedintoanRNA
moleculeiscalledatranscriptionunitand
encodesatleastonegene.
•Ifthegenetranscribedencodesforaprotein,
theresultoftranscriptionismessengerRNA
(mRNA),whichwillthenbeusedtocreate
thatproteinviatheprocessoftranslation.

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•Alternatively,thetranscribedgenemay
encodeforeitherribosomalRNA(rRNA)or
transferRNA(tRNA),othercomponentsof
theprotein-assemblyprocess,orother
ribozymes.

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ADNAtranscriptionunitencodingforaproteincontains
notonlythesequencethatwilleventuallybedirectly
translatedintotheprotein(thecodingsequence)but
alsoregulatorysequencesthatdirectandregulatethe
synthesisofthatprotein.

10. Page 10
•AsinDNAreplication,DNAisreadfrom3'→5'
duringtranscription.Meanwhile,thecomplementary
RNAiscreatedfromthe5'→3'direction.
•OnlyoneofthetwoDNAstrands,calledthetemplate
strand,isusedfortranscription.
•TheotherDNAstrandiscalledthecodingstrand,
becauseitssequenceisthesameasthenewly
createdRNAtranscript(exceptforthesubstitutionof
uracilforthymine).

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•Transcription is divided into 3
stages:
1.initiation
2.elongation
3.termination.

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Initiation
•Inbacteria,transcriptionbeginswiththebindingofRNA
polymerasetothepromoterinDNA.
•RNApolymeraseisacoreenzymeconsistingoffive
subunits:2αsubunits,1βsubunit,1β'subunit,and1ω
subunit.
•Atthestartofinitiation,thecoreenzymeisassociated
withasigmafactorthataidsinfindingtheappropriate-35
and-10basepairsdownstreamofpromotersequences.

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Elongation
•OnestrandofDNA,thetemplatestrand(ornoncoding
strand),isusedasatemplateforRNAsynthesis.
•Astranscriptionproceeds,RNApolymerasetraverses
thetemplatestrandandusesbasepairing
complementaritywiththeDNAtemplatetocreatean
RNAcopy.
•AlthoughRNApolymerasetraversesthetemplate
strandfrom3'→5',thecoding(non-template)strand
andnewly-formedRNAcanalsobeusedasreference
points,sotranscriptioncanbedescribedasoccurring
5'→3'.

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•ThisproducesanRNAmoleculefrom5'→3',anexact
copyofthecodingstrand(exceptthatthyminesare
replacedwithuracils,andthenucleotidesare
composedofaribose(5-carbon)sugarwhereDNAhas
deoxyribose(onelessoxygenatom)initssugar-
phosphatebackbone).
•UnlikeDNAreplication,mRNAtranscriptioncan
involvemultipleRNApolymerasesonasingleDNA
templateandmultipleroundsoftranscription
(amplificationofparticularmRNA),somanymRNA
moleculescanberapidlyproducedfromasinglecopy
ofagene.

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•Elongation also involves a proofreading
mechanism that can replace incorrectly
incorporated bases.

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Termination
•Bacteriausetwodifferentstrategiesfortranscription
termination:Rho-independentandRho-dependent

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Fig. Formation of
hairpin
InRho-independent
transcription
termination,RNA
transcriptionstops
whenthenewly
synthesized RNA
moleculeformsaG-C
richhairpinloop
followedbyarunof
U's,whichmakesit
detachfromtheDNA
template.

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Fig. Rho dependent
termination
Inthe"Rho-dependent"
typeoftermination,a
proteinfactorcalled
"Rho"destabilizesthe
interactionbetween
thetemplateandthe
mRNA,thusreleasing
thenewlysynthesized
mRNA from the
elongationcomplex.

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Genetic code

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Properties of genetic code
•Thecodeisuniversal.Allprokaryoticand
eukaryoticorganismsusethesamecodonto
specifyeachaminoacid.
•Thecodeistriplet.Threenucleotidesmakeone
codon.61ofthemcodeforaminoacidsand3viz.,
UAA,UAGandUGAarenonsensecodonsorchain
terminationcodons.
•Thecodeisdegenerate.Foraparticularamino
acidmorethanonewordcanbeused

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•The code is non overlapping. A base in mRNA is
not used for two different codons
•The code is commaless. There is no special signal
or commas between codons.
•The code is non ambiguous. A particular codon
will always code for the same amino acid,
wherever it is found.

22. Page 22

23. Page 23
Fig. Base sequence
of yeast alanyltRNA
Fig. Common features of
tRNAmolecule
tRNA

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Fig. 3D structure of tRNA

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Ribosomes
•Bacterialribosomesconsistsoftwosubunitsof
unequalsize,thelargerhavingasedimentation
coefficientof50Sandthesmallerof30S.
•Thetworibosomalsubunitshaveirregular
shapeswhichfittogetherinsuchawaythata
cleftisformedthroughwhichmRNApassesas
theribosomemovesalongitduringthe
translationprocessandfromwhichthenewly
formedpolypeptidechainemerges.

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Fig. Ribosomes

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Translation

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•Translationisthefirststageofprotein
biosynthesis(partoftheoverallprocessofgene
expression).
•Translationistheproductionofproteinsby
decodingmRNAproducedintranscription.
•Itoccursinthecytoplasmwheretheribosomes
arelocated.
•Ribosomesaremadeofasmallandlarge
subunitwhichsurroundsthemRNA.

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•Intranslation,messengerRNA(mRNA)isdecoded
toproduceaspecificpolypeptideaccordingtothe
rulesspecifiedbythegeneticcode.
•ThisusesanmRNAsequenceasatemplateto
guidethesynthesisofachainofaminoacidthat
formaprotein.
•ManytypesoftranscribedRNA,suchastransfer
RNA,ribosomalRNA,andsmallnuclearRNAare
notnecessarilytranslatedintoanaminoacid
sequence.

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•Translationproceedsinfourphases:
–activation,
–initiation,
–elongationand
–termination
(alldescribingthegrowthoftheaminoacid
chain,orpolypeptidethatistheproduct
oftranslation).
Aminoacidsarebroughttoribosomesand
assembledintoproteins.

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Activation
•Inactivation,thecorrectaminoacidiscovalently
bondedtothecorrecttransferRNA(tRNA).
•Whilethisisnottechnicallyastepintranslation,itis
requiredfortranslationtoproceed.
•Theaminoacidisjoinedbyitscarboxylgrouptothe3'
OHofthetRNAbyanesterbond.
•WhenthetRNAhasanaminoacidlinkedtoit,itis
termed"charged".

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Fig. Charged tRNA(N-formyl
methionyltRNA
f
met
)

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Initiation
InProkaryotesinitiationrequiresthelargeandsmall
ribosomesubunits,themRNA,theinitiatortRNAand
threeinitiationfactors(IF1,IF2,IF3)andGTP.The
overallsequenceoftheeventisasfollows
•IF3bindtothefree30Ssubunit,thishelpstoprevent
thelargesubunitbindingtoitwithoutanmRNA
moleculeandforminganinactiveribosome
•IF2complexedwithGTPandIF1thenbindstothe
smallsubunit.ItwillassistthechargedinitiatortRNA
tobind.

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•The30SsubunitattachedtoanmRNAmolecule
makinguseoftheribosomebindingsite(RBS)onthe
mRNA
•TheinitiatortRNAcanthenbindtothecomplexby
basepairingofitsanticodonwiththeAUGcodonon
mRNA.
•Atthispoint,IF3canbereleased,asitsrolein
keepingthesubunitsapartandhelpingthemRNAto
bindarecomplete.
•Thiscomplexiscalled30Sinitiationcomplex

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•The50Ssubunitcannowbind,whichdisplaceIF1
andIF2,andtheGTPishydrolysedinthisenergy
consumingstep.
•Thiscomplexiscalledas70Sinitiationcomplex.

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Fig. Formation of the 70S initiation
complex

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•TheassembledribosomehastwotRNA
bindingsites.
•ThesearecalledtheAandPsites,foramino
acylandpeptidylsites.
•TheAsiteiswhereincomingaminoacyltRNA
moleculesbind,andthePsiteiswherethe
growingpolypeptidechainisusuallyfound.
•Thesesitesareinthecleftofsmallsubunit
andcontainadjacentcodonsthatarebeing
translated.

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•Onemajoroutcomeofinitiationisthe
placementoftheinitiatortRNAintheP
site.
•ItistheonlytRNAthatdoesthis,asall
othermustentertheAsite.

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Fig. Initiation phase of protein synthesis

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Elongation
•Withtheformationof70Sinitiation
complextheelongationcyclecan
begin.
•IninvolvesthreeelongationfactorsEF-
Tu,EF-TsandEF-G,GTP,chargedtRNA
andthe70Sinitiationcomplex.

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Elongationisdividedintothreesteps:
1.AminoacyltRNAdelivery.
•EF-TuisrequiredtodelivertheaminoacyltRNAto
theAsiteandenergyisconsumedinthisstepby
thehydrolysisofGTP.
•ThereleasedEF-Tu.GDPcomplexisregenerated
withthehelpofEF-Ts.
•IntheEF-TuEF-TsexchangecycleEF-Tsdisplaces
theGDPandsubsequentlyisdisplaceditselfby
GTP.
•TheresultantEF-Tu.GTPcomplexisnowableto
bindanotheraminoacyltRNAanddeliverittothe
ribosome.
•AllaminoacyltRNAscanformthiscomplexwith
EF-TuexcepttheinitiatortRNA.

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•Fig. Elongation of polypeptide
chain

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2.Peptidebondformation.
•Afteraminoacyl-tRNAdelivery,theA-andP-sitesare
bothoccupiedandthetwoaminoacidsthataretobe
joinedareincloseproximity.
•Thepeptidyltransferaseactivityofthe50Ssubunit
cannowformapeptidebondbetweenthesetwo
aminoacidswithouttheinputofanymoreenergy,
sinceenergyintheformofATPwasusedtocharge
thetRNA.

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•Fig.Peptide bond formation

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3.Translocation.
•AcomplexofEF-G(translocase)andGTPbindsto
theribosomeand,inanenergyconsumingstep,the
dischargedtRNAisejectedfromtheP-site,the
peptidyl-tRNAismovedfromtheA-sitetotheP-
siteandthemRNAmovesbyonecodonrelativeto
onecodontotheribosome.
•GDPandEF-Garereleased,thelatterbeing
reusable.Anewcodonisnowpresentinthevacant
A-site.
Thecycleisrepeateduntiloneofthe
terminationcodons(UAA,UAGandUGA)appearin
theA-site.

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Fig. Translocation

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Termination
•TerminationofthepolypeptidehappenswhentheA
siteoftheribosomefacesastopcodon(UAA,UAG,
orUGA).
•Whenthishappens,notRNAcanrecognizeit,buta
releasingfactorcanrecognizenonsensecodonsand
causesthereleaseofthepolypeptidechain.
•The5'endofthemRNAgivesrisetotheprotein'sN-
terminus,andthedirectionoftranslationcan
thereforebestatedasN->C.

48. Page 48
Termination
•Terminationofpolypeptidesynthesisissignalled
byoneofthethreeterminationcodonsinthe
mRNA(UAA,UAGandUGA)immediately
followingthelastaminoacidcodon.
•Inprokaryotes,onceaterminationcodon
occupiestheribosomalA-site,threetermination
orreleasefactors,viz.,theproteinRF1,RF2and
RF3contributeto-
Thehydrolysisoftheterminalpeptidyl-tRNA
bond.
Releaseofthefreepolypeptideandthelast
unchargedtRNAfromtheP-site
Thedissociationofthe70Sribosomeintoits30S
and50Ssubunits

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•RF1recognizestheterminationcodonUAG
andUAAandRF2recognizeUGAandUAA.
•EitherRF1orRF2bindsatthetermination
codonandinducespeptidyltransferaseto
transferthegrowingpeptidechaintoa
watermoleculeratherthantoanother
aminoacid.
•FunctionofRF3isnotknown.

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Fig. Termination

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Polyribosomes
•AsinglestrandofmRNAistranslated
simultaneouslybymanyribosomes,spaced
closelytogether.
•Suchclustersofribosomesarecalled
polysomesorpolyribosomes.
•Thesimultaneoustranslationofasingle
mRNAbymanyribosomesallowhighly
efficientuseofthemRNA

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Fig. Polyribosomes

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