(6.5.15)--Structural-Basis-of-Transcriptio.pdf
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1、Cell,Vol.119,481489,November 12,2004,Copyright 2004 by Cell PressStructural Basis of Transcription:Nucleotide Selection by Rotationin the RNA Polymerase II Active CenternucleotidetotheAsite).Inthestructureofthistranscrib-ingcomplex,theDNAbase ati?1iscontacted byaminoacid side chains of a structural
2、element termed the“bridge helix.”In pol II,the bridge helix is essentiallystraight,whereas in the structure of bacterial RNA poly-Kenneth D.Westover,David A.Bushnell,and Roger D.Kornberg*Department of Structural BiologyStanford University School of MedicineStanford,California 94305merase(Zhang et al
3、.,1999),the bridge helix is bent,placing the corresponding amino acid side chains inposition to contact the DNA base at position i?2.ThisSummaryobservation led to the proposal that transitions betweenstraight and bent states of the bridge helix underlie theBinding of a ribonucleoside triphosphate to
4、 an RNApolymerase II transcribing complex,with base pairingtranslocation step in transcription(Cramer et al.,2001).Evidence in support of this hypothesis has come fromtothetemplateDNA,wasrevealedbyX-raycrystallog-raphy.Binding of a mismatched nucleoside triphos-RNA-protein crosslinking studies,in wh
5、ich crosslinkscharacteristic of both straight and bent states of thephate was also detected,but in an adjacent site,in-verted with respect to the correctly paired nucleotide.bacterial enzyme were observed(Epshtein et al.,2002).A second structure of a pol II transcribing complexThe results are consis
6、tent with a two-step mechanismof nucleotide selection,with initial binding to an entrywas obtained with the use of RNA formed not by tran-scription,but rather as a synthetic oligonucleotide(E)sitebeneaththeactivecenterinaninvertedorienta-tion,followed by rotation into the nucleotide addition(Westove
7、r et al.,2004).An eight-or nine-residue RNAand complementary strand of DNA form a stable com-(A)site for pairing with the template DNA.This mecha-nism is unrelated to that of single subunit RNA poly-plex with pol II(Kireeva et al.,2000).A chain terminating3?-deoxyadenylate residue was added by trans
8、cription.merases and so defines a new paradigm for the large,multisubunit enzymes.Additional findings from theseThe structure of this transcribing complex revealed avacant A site and therefore represented the“post-studiesincludeathirdnucleotidebindingsitethatmaydefine the length of backtracked RNA;D
9、NA doubletranslocation”state.The structure further revealed theunwinding of the DNA-RNA hybrid at the upstream endhelix unwinding in advance of the polymerase activecenter;and extension of the diffraction limit of RNAof the RNA,involving a set of protein loops in a networkof protein-nucleic acid int
10、eractions.polymerase II crystals to 2.3 A.Wehave nowexploitedtheformation ofatranscribingcomplexintheposttranslocationstatetoinvestigatetheIntroductionmode of interaction with substrate NTP.Transcribingcomplex crystals were soaked with both NTP matchedThe elementary step in transcription may be subd
11、ividedinto multiple stages:selection of a ribonucleoside tri-to the DNA base at position i?1 and unmatched NTPs.The resultingstructures revealed anunexpected featurephosphate complementary to the DNA template;cataly-sisofphosphodiesterbondformation;andtranslocationof the nucleotide entry and additio
12、n mechanism.of the RNA and DNA,with concomitant unwinding ofthe RNA-DNA hybrid helix and unwinding and rewindingResultsof the DNA double helix.Previous X-ray crystal struc-tures of yeast RNA polymerase II(pol II)transcribingWe previously formed a transcribing complex throughcomplexes have given insi
13、ght into the mechanisms ofthe binding to pol II of a nine-residue RNA oligonucleo-translocation and helix unwinding(Gnatt et al.,2001;tide and a 15-residue DNA oligonucleotide containingWestover et al.,2004).We now report structures of tran-complementary sequence(Westover et al.,2004).Wescribingcomp
14、lexesthatareinformativeaboutthemech-have now extended the downstream region of the tem-anisms of nucleotide selection and catalysis.plate strand with 14 residues of duplex DNA(Figure 1),In the first structure of a pol II transcribing compleximproving the reproducibility and size of the crystals(Gnat
15、tetal.,2001),transcriptionwasstalledbytheomis-and their stability to manipulation before freezing.Intro-sion of a nucleoside triphosphate(NTP).The structureduction of a noncomplementary NTP before freezingrevealed the last nucleotide added to the RNA,still ingave the best diffraction data.Structures
16、 were solvedthe“nucleotide addition”(here designated“A”)site,op-by molecular replacement with an earlier transcribingposite the DNA base at position i?1 in the templatecomplex model(Gnatt et al.,2001)and rigid body refine-strand.The structure therefore represented the“pre-ment(Table 1).translocation
17、”state(although the complex must haveundergone translocation,advancing the last nucleotideDNA Unwinding in the Transcribing Complexadded and the associated DNA base to position i?2,As with the previous transcribing complex formed fromexposing the A site to create a requirement for the miss-RNAandDNA
18、oligonucleotides,thepresenttranscribinging NTP,and stalling transcription;the complex mustcomplex was in the posttranslocation state.The struc-then have undergone backtracking to return the lastturedifferedfromthoseobtainedpreviouslybythepres-ence of connected density for the template strand be-yond
19、 the downstream end of the DNA-RNA hybrid*Correspondence:kornbergstanford.eduCell482Figure 1.DNA and RNA in the Structure of a Pol II Transcribing Complex(A)Model for DNA and RNA fitted to electron density for nucleic acids(2Fo?Fcmap,with phases from pol II alone,contoured at 0.8?).Viewis the same a
20、s Figure 1 of Westover et al.(2004).Color code is at upper right.A chain-terminating 3?dA residue is shown in yellow.(B)Sequences of DNA and RNA in the transcribing complex.Color code as in(A).Nucleotide positions are numbered with respect to theaddition site at?1(denoted i?1 site),with positions up
21、stream extending from?1 and those downstream from?2.Separation of DNA andRNA strands upstream of?8 and separation of DNA strands upstream of?6 are shown schematically.Figures were generated by PyMOLor SPOCK.(Figure 1).The downstream double-stranded DNA re-the RNA,could be soaked in Mn-or Mg-UTP,comp
22、le-mentary to the DNA base at position i?1,without addi-gion was also better defined,lying on the Rpb1 side ofthe active center cleft(not shown).Beyond the templatetion of U to the RNA.Diffraction data were collectedto 4.2 Aresolution(Table 1),and a difference electronresidue at i?1,additional unpai
23、red bases were resolvedon the template strand at the downstream positionsdensitymapwascalculatedbysubtractingthestructurewith the DNA and RNA removed(2Fo?Fcomit map).The?2,?3,and?4.The next base on the template strandat position?5 was also unpaired,despite the presencedifference map showed density f
24、or UTP in the A site(Figure 2A),with the nucleotide base paired to the adja-of a complementary base in the nontemplate strand.This“fraying”of the end of the downstream duplex wascent template DNA base and with the?phosphate posi-tioned for in-line nucleophilic attack by an OH group atmaintained by i
25、nteraction of the nontemplate residue atposition?5 with Rpb1 residues Lys1109 and Asn1110the 3?end of the RNA(absent from the chain-termi-nated RNA).in the floor of the active center cleft.Fraying of theend may have been due,in part,to the absence of anyAn anomalous difference map obtained from a cr
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