核糖体开关的研究进展

核糖体开关的研究进展第1页/共46页T7RNApolymerase-catalyzedinvitrorun-offtranscriptionsystemTheRNAtohaveapurinerichsequenceatthe5'terminus.ThesubsequentRNAisheterogenousinlengthduetorun-offtranscription,T7RNApolymerasetendstoaddextranucleotidestothe3-endofthedesiredRNA.第2页/共46页第3页/共46页RibozymeRibozymesareantisenseRNAmoleculesthathavecatalyticactivity.TheyfunctionbybindingtothetargetRNAmoietythroughWatson-Crickbasepairingandinactivateitbycleavingthephosphodiesterbackboneataspecificcuttingsite.Theymaycatalyzeself-cleavage(intramolecularor"in-cis"catalysis)aswellasthecleavageofexternalsubstrates(intermolecularor"in-trans"catalysis)HammerheadribozymeHepatitisdeltavirusribozymeHairpinribozymeVarkudsatellite(VS)ribozyme

第4页/共46页ThehairpinribozymeThenameofhammerheadribozymeisgivenbythesimilaritybetweenitssecondarystructureandtheshapeofahammerheadThehammerheadribozymeisanantisenseRNA.SomeoftheribonucleotideswithinthesequenceselectivelyformWatson-Crickbasepairswithotherstoformastem,whilethereststayinsinglestrandedstatecalledloop.第5页/共46页TurnovercycleofRNAcleavagebyahammerheadribozymeBothofturnoverandspecificityareaffectedbybindingarmlength(helixIandIII).Ifthelengthofbindingarmsisveryshort,therateofdissociationofthetargetfromtheribozymemayexceedtherateofcleavage,resultinginpoorefficiency(Rossi,1997).However,stablehybridsexhibitlowcatalyticactivitybecauseofslowdissociationofthecleavedsubstrate(Bertrand,1994).Thus,theidealsituationistohavearmlengthsthataidcleavage,yetprovideforquickdissociationofthecleavedproducts.第6页/共46页Useofcis-andtrans-ribozymestoremove5’and3’heterogeneitiesfrommilligramsofinvitrotranscribedRNA1.sequencerequirementsforcleavageimmediatelyupstreamofthecleavagesitemaybeapproximatedtoaNUX(N=rA,rC,rGorrU,X=rA,rCorrU)trinucleotide.2.Bothofturnoverandspecificityareaffectedbybindingarmlength(helixIandIII).Ifthelengthofbindingarmsisveryshort,therateofdissociationofthetargetfromtheribozymemayexceedtherateofcleavage,resultinginpoorefficiency(Rossi,1997).However,stablehybridsexhibitlowcatalyticactivitybecauseofslowdissociationofthecleavedsubstrate(Bertrand,1994).Thus,theidealsituationistohavearmlengthsthataidcleavage,yetprovideforquickdissociationofthecleavedproducts.3.DivalentcationsarebelievedtoparticipateinthecleavagereactionwithMg2+andMn~beingpreferred第7页/共46页Notethatthefirst11ntofdomainVarecomplementarytoaportionofHHNucleicAcidsResearch,1996,Vol.24,No.5977–978第8页/共46页Varkudsatellite(VS)ribozymeHowever,sometimesthewell-characterizedhammerheadhassequencerequirements5’tothecleavagesitethatareincompatiblewithdesiredproduct.第9页/共46页Varkudsatellite(VS)ribozymeThehammerheadribozyme(HH)andVSribozymesubstratestem–loop(VSsl)areinlowercase.Bondsthatarecleavedbytheribozymesareshowninbrokenlines.第10页/共46页Productionofhomogeneousd56RNAwithuseofcis-andtrans-ribozymesLaneA,d56;laneB,d56-d;laneC,d56-VSslintheabsenceofVSribozyme;laneD,asCwithVSribozymeadded;laneE,HH-d56-VSslwithoutVSribozyme;laneF,sameasE,withVSadded.Transcriptionreactions(10mleach)contained50mg/mllinearizedplasmidDNA第11页/共46页HepatitisdeltavirusribozymeThehepatitisdeltavirus(HDV)ribozymeisanon-codingRNAthatisnecessaryforviralreplicationandisthoughttobetheonlycatalyticRNAknowntoberequiredforviabilityofahumanpathogen.TheribozymeactstoprocesstheRNAtranscriptstounitlengthsinaself-cleavagereactionRNA(2004),10:988–995.第12页/共46页5’-AGCCGGCCATGGTCCCAGCCTCCTCGCTGGCGGCCGGTGGGCAACATCGTTCGCGATGGCGAATGGGACC第13页/共46页第14页/共46页第15页/共46页第16页/共46页WorkprogressLGR5NASPandhistones第17页/共46页AdultStemCell-DrivenEpithelialRenewalintheSmallIntestine第18页/共46页Wntsignalingpathway第19页/共46页SchematicrepresentationoffourhumanRSPOproteinsHSPG:heparinsulfateproteoglycans肝素蛋白多糖第20页/共46页Cristin/R-spondinsaresecretedproteinsitispredictedthatHSPGsaffectcellsurfacebindingofRSPO,theadditionofsolubleheparin(asugarmoietysimilartotheonefoundonHSPGs)orsodiumchlorate(aninhibitorofsulfatasesmediatingsulfationofHSPGs)tothemediumduringcultureofcellsoverexpressingRspossignificantlyenhancedthedetectionofsecretedRSPOproteinsintheconditionedmediumFurthermore,deletionoftheTSRdomainfromRSPOsresultedinanincreaseofsolubleRSPOproteinsinconditionedmedium第21页/共46页第22页/共46页FurindomainsconfertherelativeactivityofRSpoproteins第23页/共46页Whole-CellBindingAnalysisR-spondinsfunctionasligandsoftheorphanreceptorsLGR4andLGR5第24页/共46页TheN-terminaldomainsofLGR5arerequiredforRSPO1bindingΔN:DeletionoftheextremeN-terminalofLGR5(28-50)ΔLRR1:Δ(51-75)ΔLRR1-17第25页/共46页第26页/共46页第27页/共46页Vectormodification

Fc-9*HIS第28页/共46页LGR-VLRchimeraconstructspFastbacHTA-gp67-2Fc-9HispFastbacHTA-gp67-Lgr5(27-163)-VLR-2Fc-9HispFastbacHTA-gp67-Lgr5(27-211)-VLR-2Fc-9HispFastbacHTA-gp67-Lgr5(27-258)-VLR-2Fc-9HispFastbacHTA-gp67-Lgr5(27-353)-VLR-2Fc-9HispFastbacHTA-gp67-Lgr5(27-399)-VLR-2Fc-9His第29页/共46页lkelaldtnqlksvpdgifdrltslqkiwlhtnpwdcscpridylsrwlnknsqkeqgsakcsgsgkpvrsiicpt第30页/共46页lkelaldtnqlksvpdgifdrltslqkiwlhtnpwdcscpridylsrwlnknsqkeqgsakcsgsgkpvrsiicpt第31页/共46页pFastbacHTA-gp67A-RSPOpFastbac-gp67A-RSPO1(34-135)pFastbac-gp67A-RSPO1(34-208)pFastbac-gp67A-RSPO2(22-148)pFastbac-gp67A-RSPO4(33-137)pFastbac-gp67A-RSPO4(33-199)pFastBac-GST-RSPO1(34-135)pFastBac-GST-RSPO1(34-208)pFastBac-GST-RSPO2(22-148)pFastBac-GST-RSPO4(33-137)pFastBac-GST-RSPO4(33-199)第32页/共46页pFastbacHTA-gp67A-RSPO-2Fc-9HispFastbac-gp67A-RSPO1(34-135)-Fc-HispFastbac-gp67A-RSPO1(34-208)-Fc-HispFastbac-gp67A-RSPO2(22-148)-Fc-HispFastbac-gp67A-RSPO2(22-205)-Fc-His第33页/共46页pFastbacDual-RSPO-LGR5pFastBacdual-Rspo1-LGR5(1-455)-6*HISpFastBacdual-Rspo1-LGR5(1-464)-6*HISpFastBacdual-Rspo1-LGR5(1-434)-6*HISpFastBacdual-Rspo1-LGR5(1-447)-6*HISMouseLGR5andLGR4第34页/共46页NASPMaintainsaSolubleH3-H4PoolbyProtectingThemfromDegradationbyCMA第35页/共46页NASPInteractswiththeNTerminusofH3第36页/共46页NASP(26-290)35kd25kdNasp(26-290)Nasp(38-338)Nasp(26-290)第37页/共46页2012-12-13NASP(26-290)Superdex75Peak:750mUV8mlCond显示20ms/cm，估计盐浓度大概在300左右，buffer配置有问题？Superdex75如果蛋白分子量大于75kd会穿出，8毫升左右代表穿出，但是不知道是几聚合体，明天过superdex200看看是几聚合体第38页/共46页NASP(26290)Superdex200prep第39页/共46页NASP(26290)Hiload26/260200