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©2014PearsonEducation,Inc.Chapter8RegulatoryRNAs•RegulationbyRNAsinBacteria•RegulatoryRNAsareWidespreadinEukaryotes•SynthesisandFunctionofmiRNAMolecules•SilencingGeneExpressionbySmallRNAs•LongNon-CodingRNAsandX-Inactivation©2014PearsonEducation,Inc.RegulatoryRNAs•Inrecentyears,therehasbeenanexplosioninthestudyofRNAregulators,particularlyineukaryotes,thatoperateattheleveloftranscriptionandespeciallytranslation.•Thisnewfieldemergedfromtwosources:thediscoveryofmicroRNAs,firstreportedintheearly1990s,andthenthediscoveryofthephenomenonknownasRNAinterferenceinthelate1990s.•Beforewedescribetheseformsofregulation—howtheyworkandtheapplicationstheyaffordresearchers—weconsidercasesofRNA-mediatedgeneregulationinbacteria.©2014PearsonEducation,Inc.REGULATIONBYRNAsINBACTERIA•SmallRNAshavebeenrecognizedinprokaryotesformanyyears.Someareinvolvedinregulatingthereplicationofplasmids,andothersareinvolvedinregulatinggeneexpression.•SomeoftheseRNAscontroltranscription—the6SRNAofE.coli,forexample.ThisRNAbindstotheσ70subunitofRNApolymeraseanddown-regulatestranscriptionfrommanyσ70promoters.•The6SRNAaccumulatesathighlevelsinstationaryphase.Instationaryphase,analternativeσfactor,σS,ismade.Thisσcompeteswithσ70forcorepolymeraseanddirectstheenzymetopromotersexpressinggenesforthemultiplestressresponsesneededtosurvivestationaryphase.Bydown-regulatingtranscriptionfromσ70promoters,6SRNAhelpsthisshiftinexpressiontoσSpromoters.•Inrecentyears,attentionhasfocusedonsmallRNAmoleculesinbacteriathatregulatetranslationandmRNAdegradation.©2014PearsonEducation,Inc.•OneclassofbacterialregulatoryRNAs(calledsRNAs)actsintranstocontroltranslationoftargetgenes,ratherasmicroRNAsdoineukaryotes.•Theyare,however,larger(80–110nt)thanthoseeukaryoticregulatoryRNAs(21-30nt),andtheyarenotgenerallyformedbyprocessingoflargerdouble-strandedRNA(dsRNA)precursors(asthoseeukaryoticRNAregulatorsare);instead,theyareencodedintheirfinalformbysmallgenes.•Manyofthesegeneshavebeenidentifiedbybioinformatics,withmorethan100sRNAsbeinguncoveredinE.coli.•MostsRNAsworkbybasepairingwithcomplementarysequenceswithintargetmRNAsanddirectingdestructionofthemRNA,inhibitingitstranslationoreveninsomecasesstimulatingtranslation.©2014PearsonEducation,Inc.Hfqprotein•BindingofansRNAtoitstargetmRNAisinmostcasesaidedbythebacterialproteinHfq.•ThisRNAchaperoneisneededbecausethecomplementaritybetweenthesRNAsandtheirtargetmRNAsistypicallyimperfectandshort,andthustheirinteractionisweak.•Hfqfacilitatesbasepairing.Also,bybindingthesRNAsevenbeforetheyarepairedwiththeirtargets,Hfqincreasesthestabilityoftheseregulators.©2014PearsonEducation,Inc.RybBRNA•Awell-studiedsRNAfromE.coliisthe81-nucleotideRybBRNA.ThissRNAbindsseveraltargetmRNAsandtriggerstheirdestructionbecausethedouble-strandstretchofheteroduplexformeduponpairingisrecognizedasasubstratebythenucleaseRNaseE.•MostofthemRNAstargetedbyRybBencodeironstorageproteins.Freeironisrequiredbythecellundercertaincircumstances,buthighlevelsaretoxic.RybBregulatesthelevelsoffreeironbycontrollingthelevelsofironstorageproteins.•RybBisexpressedfromapromoterrecognizedbyaspecialσfactorcalledσE.ExpressionofthegeneencodingσEisitselfregulatedbyRybB,andthusthissRNAispartofanautonegativeregulatoryloopforσE.©2014PearsonEducation,Inc.Figure20-1ActivationandrepressionoftranslationbysRNAs©2014PearsonEducation,Inc.RiboswitchesResidewithintheTranscriptsofGenesWhoseExpressionTheyControlthroughChangesinSecondaryStructure•Riboswitchescontrolgeneexpressioninresponsetochangesintheconcentrationsofsmallmolecules.•Theseregulatoryelementsaretypicallyfoundwithinthe5′-untranslatedregions(5′-UTRs)ofthegenestheycontrol,andtheycanregulateexpressionattheleveloftranscriptionortranslation.•TheydothisthroughchangesinRNAsecondarystructure.©2014PearsonEducation,Inc.Figure20-2Eachriboswitchismadeupoftwocomponents:theaptamerandtheexpressionplatform.Theaptamerbindsthesmall-moleculeligandand,inresponse,undergoesaconformationalchange,which,inturn,causesachangeinthesecondarystructureoftheadjoiningexpressionplatform.Theseconformationalchangesalterexpressionoftheassociatedgenebyeitherterminatingtranscriptionorinhibitingtheinitiationoftranslation.适配体表达平台©2014PearsonEducation,Inc.•InBacillussubtilis,manygenesinvolvedintheuseoftheaminoacidmethioninehavea200-nucleotide-longuntranslatedleaderRNAthatactsasaSAM(S-adenosylmethionine,S-腺苷甲硫氨酸)–sensingriboswitch.•RNApolymeraseinitiatestranscriptionatthepromoterandtranscribesthroughthisleaderregionbeforeenteringthecodingsequenceofthedownstreamgenes.OncetranscribedintoRNA,theleaderregioncanadoptalternativestructuresthroughalternativepatternsofintramolecularbasepairing.©2014PearsonEducation,Inc.•Onearrangementincludesastem-looptranscriptionalterminator.SAM—theligandforthisriboswitch—bindstotheaptamerandstabilizesthesecondarystructurethatincludesthistranscriptionalterminator.Underthesecircumstances,transcriptionisterminatedbeforepolymerasehasachancetotranscribethedownstreamprotein-codingsegmentofthegene.•Thisformoftranscriptionalregulationisalsocalledattenuation.©2014PearsonEdu