12Mechanisms of Transcription.docx

12Mechanisms of Transcription.docx

《12Mechanisms of Transcription.docx》由会员分享，可在线阅读，更多相关《12Mechanisms of Transcription.docx（20页珍藏版）》请在冰豆网上搜索。

12MechanismsofTranscription

MechanismsofTranscription

Chapter12

CentraltoourunderstandingofmolecularbiologyisthatthegeneticinformationcontainedinDNAiscopiedintoRNA.

Thisprocessofcopyingiscalledtranscription.

A.Fourrequirementsfortranscription

B.Stepsintranscription

C.TranscriptioninEukaryotes

A.Fourrequirementsfortranscription

Thereare4thingsthatarenecessaryfortranscriptionofDNA:

1.templateDNA

2.RNApolymerase

3.promoter

4.4rNTPs

1.templateDNA

DNAisdoublestrandedbutonlyonepart

ononestrandistranscribedatatime.

ThisisthetemplateDNA.

ThetemplateDNAdeterminesthesequenceoftheribonucleotidebasesintheRNA.

WhatisthenamefortheenzymethatcopiesDNAintoRNA?

RNApolymerase

2.RNAPolymerase

ThestructureofRNApolymerasehasbeendeterminedforseveralspecies.

DNApolymerasewasfoundtobeshapedlikeahand.

RNApolymeraseisshapedlikeacrabclaw.

Whatisacrab?

2.RNAPolymerase

Thereare2formsofbacterialRNApolymerasesbasedontheproteinsubunitsthatarepresent:

a.thecoreenzyme

b.theholoenzyme

Whatisaholoenzyme?

Aholoenzymeisacomplexofmanysubunitsthathasacoreenzyme,whichhasfunction,butthisfunctionisincreasedbythepresenceofotherproteinsubunits.

a.thecoreenzyme

TheRNApolymerasecoreenzymeismadeupof4differentsubunits:

1.twoα（alpha）subunits

2.oneβ（beta）subunit

3.oneβ’（betaprime）subunit

4.oneω（omega）subunit

a.thecoreenzyme

ThecoreenzymecanbindDNAandtranscribeitintoRNA,butithaslessactivitythantheholoenzyme.

ThecoreenzymebeginstranscriptionatrandompointsintheDNA（itdoesnotrecognizepromotersliketheholoenzyme.）

UnlikeDNApolymerase,noprimerisrequiredbyRNApolymerase.

TheactivesiteofRNApolymeraseisatthebaseoftheclaw.SimilartoDNApolymerase,theactivesitecanbind2Mg++ions.

b.theholoenzyme

Theholoenzymehasthe4subunitsfromthecoreenzymeplusσ（sigma）.

σ（sigma）actsasaspecificityfactor.ItspecificallybindstopromotersequencesintheDNA.Ithas2α（alpha）helicesthatbindinthemajorgroove.InE.colithemainσfactorisnamedσ70.

Whatarepromotersequences?

Apromotersequenceisasignalwheretranscriptionshouldstart.

AttheendofthegeneadifferentDNAsequencesignalsthattranscriptionshouldstop.Thisiscalledtheterminator.

Thesidechainsonsigma70bindspecificallytocertainDNAbasepairsatthepromoter.

TheholoenzymebindstotheDNAandthenslidesalongtheDNAuntilitfindsapromotersequence.

3.Promoter

Thesigmapartoftheholoenzymerecognizesaspecificsequencecalledapromoterandthisiswheretranscriptionbegins.

3.promoter

WhatisdifferentabouttheDNAsequenceatthepromoterthatisrecognizedbytheσsubunitoftheholoenzyme?

ScientistssequencedtheDNAatmanydifferentpromotersandcomparedthesequences.Thereweresomebasepairsthatwerepresentatthesameplaceinnearlyeverypromoter.

Theotherconsensussequenceisat-35basepairsfromthestartsiteandiscalledthe-35regionor-35element.

AconsensussequenceisasequenceofnucleotidesoraminoacidsincommonbetweenregionsofhomologyindifferentbutrelatedDNAorRNAorproteinsequences.Oftenthesequencesofmanydifferentgenesorspeciesarecomparedtoidentifytheconsensussequence.

StrongpromotershaveDNAsequencesthatarecloseoridenticaltotheconsensussequenceforthe-10and-35boxes.

Promotersthatdifferfromtheconsensussequenceswillbeweaker---therewillbelesstranscriptionofthegene.

Besidesthe-10and-35regionssomestrongpromotersalsohaveaUPelement.

ThissequenceisalsorecognizedbytheRNApolymerase.Itcanincreasetranscriptionofageneby30times.

Somepromoterslacka-35regionandinsteadhaveanextended-10region.

RecentlyscientistshaveidentifiedanewelementthatRNApolymerasebindsto.

Thissequenceiscalledthediscriminatoranditislocateddownstreamofthe-10region.

4.4rNTPs

InDNAreplication,adeoxynucleotidebasepairstothenucleotideonthetemplatestrand.ThenaphosphodiesterbondisformedbyDNApolymerase.

TranscriptionissimilartoDNAreplicationinthatthenewbase,whichinthiscaseisaribonucleotide,basepairstothenucleotideonthetemplatestrand.

RNApolymeraseformsaphosphodiesterbondtoattachthenewbasetothegrowingRNA.

Aphosphodiesterbondformsbetweenthe3’hydroxylgroupandthephosphateonthenewbasepair.

Intranscriptionthenewbasepairisaribonucleicacid:

rATP,rUTP,rGTP,rCTP

Apyrophosphateisreleasedafterthephosphodiesterbondisformed.

Summaryofrequirementsfortranscription

A.Fourrequirementsfortranscription:

1.templateDNA

2.RNApolymerase

3.promoter

4.4rNTPs

Summaryofrequirementsfortranscription

1.templateDNA:

DNAthatencodesRNAandmaybeoneitherstrand.

2.RNApolymerase

a.thecoreenzymehas4differentsubunits:

1.twoα（alpha）subunits

2.oneβ（beta）subunit

3.oneβ’（betaprime）subunit

4.oneω（omega）subunit

2.RNApolymerase

b.theholoenzymehas5differentsubunits:

1.twoα（alpha）subunits

2.oneβ（beta）subunit

3.oneβ’（betaprime）subunit

4.oneω（omega）subunit

5.oneσ（sigma）subunit

σ（sigma）specificallyrecognizespromoterDNA.

3.promoter

Scientistsidentifiedthepromoterconsensussequence.

Thereisa-10regionanda-35region.

SomepromoterssometimesalsohaveaUPelement.Othershaveanextended-10boxoradiscriminator.

4.4rNTPs:

rATP,rUTP,rGTP,rCTP

B.StepsinTranscription

Therearethreestepsintranscription.

Whatarethey?

1.Initiation

2.Elongation

3.Termination

1.Initiation

a.Thereare3stepsininitiation:

1.formingtheclosedcomplex

2.formingtheopencomplex

3.additionofthefirstnucleotidesandpromoterescape（lossofsigma）

1.formingtheclosedcomplex

RNApolymerasebindstothepromotersequence.

Thisiscalledtheclosedcomplex.

1.formingtheclosedpromotercomplex

RNApolymerase（holoenzyme）bindsnonspecificallytoDNAuntilitfindsapromoter.SpecificbindingoccursatpromotersandtheDNAisboundbyσ.

WhattechniquecouldscientistsusetodeterminewhatDNAbasepairsareboundbyRNApolymerase?

nucleaseprotectionfootprinting

Nucleaseprotectionfootprinting

ThelocationofproteinbindingsitesonDNAcanbedeterminedbynucleaseprotectionfootprinting.

TheproteinisboundtotheDNA.ThenDNAisdigestedwithDNases,butthepartthattheproteinwasboundtoisprotectedfromdigestion.

Aradioactivemolecule（32P）isaddedtotheendoftheDNApieces.

ThentheDNAisseparateonagel.Thegelisplacedagainstapieceofphotographicfilm.Wherethereisradioactivity,thefilmwillturnblack.Thisiscalledautoradiography.ItisusedtovisualizetheDNAbands.

Basedonnucleaseprotectionfootprintingexperiments,itwasshownthatRNApolymerasebindsfrom-44to+2ontheDNA.

WhichsubunitofRNApolymerasebindstoUPelements?

Howcouldwedeterminethis?

ThepartoftheαsubunitthatbindstotheUPelementiscalledαCTD（CTDstandsforcarboxylterminaldomain）.Aflexiblelinkerconnectsthistotherestoftheαsubunit.TheotherpartoftheαsubunitiscalledNTDforaminoterminaldomain.

PartoftheαsubunitcalledtheαCTDbindstotheUP-element.

InitiallytheRNApolymerasebindsweaklytotheDNApromoter,formingtheclosedpromotercomplex.

Thenitbindsmoretightlyandformstheopencomplex.

2.formingtheopencomplex

TheDNAstrandsofthedoublehelixseparate.About14basepairsaroundthesiteoftheinitiationoftranscriptionopentoformatranscriptionbubble.Thisiscalledtheopencomplex.

2.formingtheopencomplex

AftertheRNApolymeraseisboundtoapromoter,thehydrogenbondsbetweensomebasesarebroken,openinguptheDNAfromaroundbasepairs-10to+3.Thisiscalledanopencomplex.ThisDNAisusuallyA-Trich,whichmakesdenaturingtheDNAeasier.Why?

3.additionofthefirstnucleotidesandpromoterescape

OncetheDNAisdenatured,theRNApolymerasehasaccesstotheDNAtemplateandcanbeginaddingbasepairs.RNApolymerasedoesnotrequireaprimerlikeDNApolymerase.Atthispointthecomplexiscalledtheinitialtranscribingcomplex.

Thefirstbasepairthatisaddedisusuallyanadenine.TheRNApolymerasebindstotheadenineandholdsitinplaceforanotherribonucleotidetocomeandbeboundtoit.

RNApolymeraseformsaphosphodiesterbondbetweentheadenineandthenextnucleotide.Thenmorenucleotidesareadded.

ThiscomplexisnotstableandtheRNApolymeraseoftenreleasesthetemplateduringthefirst10ribonucleotides.Thenitwilloftenbindandbeginagain.

ShortRNAmoleculesoflessthan10basepairsareproducedandreleasedbytheRNApolymerase.ThentheRNApolymerasebeginsagainwithoutdissociatingfromtheDNAtemplate.

promoterescape（lossofsigma）

Afterabout10nucleotidesareadded,theRNApolymerasebeginstomoveforwardonthetemplateDNA.Theσ（sigma）partoftheholoenzymedissociates.Thisiscalledpromoterescape.Afterpromoterescape,theelongationpartoftranscriptionbegins.

Theσ（sigma）moleculewillbindtoanotherRNApolymerasemoleculeandbegininitiationagain.

Becauseitisusedmanytimes,wesayitisrecycled.

Theσ70factormakesspecificcontactswiththeDNAatthe-10regionand-35region.

Theσ70factorisdividedinto4regions.Region4formsahelix-turn-helixbindingmotifthatbindstotheDNAatthe-35element.

Theσ70factorbindstothe-35regionwithahelix-turn-helixmotif.

Theσ