10620.full - Proc.Natl.Acad. Sci.USA...

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Unformatted text preview: Proc.Natl.Acad. Sci.USA Vol.93,pp.10620-10625,October1996 Biochemistry High-resolutionmapping of nucleoprotein complexes by site-specificprotein-DNA photocrosslinking: Organization of the human TBP-TFIIA-TFIIB-DNA quaternarycomplex THIERRYLAGRANGE*, TAE-KYUNG KIM*, GEORGE ORPHANIDES*, YON W. EBRIGHTt, RICHARD H.EBRIGHTt, AND DANNY REINBERG*t *Howard HughesMedicalInstitute,Department of Biochemistry,DivisionofNucleicAcidEnzymology,UniversityofMedicineandDentistryof New Jersey, RobertWood Johnson MedicalSchool,Piscataway,NJ 08854;and tDepartmentofChemistryandWaksman Institute,RutgersUniversity,New Brunswick, NJ 08855 Communicated byKeithR. Yamamoto, UniversityofCalifornia,San Francisco, CA,July11,1996 (receivedforreviewMay 7,1996) ABSTRACT We have used a novel site-specificprotein- DNA photocrosslinking procedure todefinethepositions of polypeptidechains relativetopromoter DNA inbinary,ter- nary, and quaternary complexes containing human TATA- binding protein, human or yeast transcription factor IIA (TFIIA), human transcription factorIIB (TFIIB), and pro- moterDNA. TheresultsindicatethatTFIIAandTFIIBmake more extensive interactionswith promoter DNA than previ- ouslyanticipated.TATA-binding protein,TFHA, and TFIIB surroundpromoterDNA fortwoturnsofDNA helixandthus may form a "cylindrical clamp" effectively topologically linkedtopromoterDNA.Ourresultshaveimplicationsforthe energetics,DNA-sequence-specificity, and pathwayofassem- blyofeukaryotictranscriptioncomplexes. Transcriptioninitiationata eukaryoticprotein-encodinggene requiresassemblyonpromoterDNA ofatranscriptioncomplex consistingofRNA polymerase IIand sixgeneraltranscription factors:TFIIA,TFIIB,TFIID[orTATA-bindingprotein(TBP)], TFIIE,TFIIF,andTFIIH(1).Thesmallesttranscriptioncomplex generallycompetent forbasaland activatedtranscriptioncon- tainsatleast35distinctpolypeptidesandhasamolecularmassin excessof2MDa (1).Understandingtranscriptioninitiationand transcriptionactivationwillrequireelucidationofthestructures andthearrangementofthesepolypeptidesrelativetoeachother andtopromoterDNA. Recently,structureshavebeendeterminedforseveralpolypep- tidesandpolypeptidedomainswithinthetranscriptioncomplex: i.e.,TBP core domain (TBPc), alone and inthe TBPc-DNA binarycomplex(2-6);TFIIAintheTBPc-TFIIA-DNA ternary complex(7,8);TFIIB coredomain (TFIIBc),aloneandinthe TBPc-TFIIBc-DNA ternary complex (9, 10); and TFIIB N- terminaldomain(11).However,thefullyassembledtranscription complexistoolargeforstructuredeterminationbyNMR spec- troscopicandx-raycrystallographicmethods.Therefore,infor- mation regardingthe arrangement ofpolypeptides relativeto each other and topromoter DNA within thefullyassembled transcriptioncomplex mustcomefromimaging (12)orbiochem- icalmethods (13-15)....
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10620.full - Proc.Natl.Acad. Sci.USA...

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