Time-Resolved Events on the Reaction Pathway of Transcript Initiation by a Single-Subunit RNA Polymerase: Raman Crystallographic Evidence.
The nucleotidyl transfer reaction leading to formation of the first phosphodiester bond has been followed in real time by Raman microscopy, as it proceeds in single crystals of the N4 phage virion RNA polymerase (RNAP). The reaction is initiated by soaking nucleoside triphosphate (NTP) substrates an...
| Publicado en: | Journal of the American Chemical Society Vol. 133; no. 32; pp. 12544 - 12556 |
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| Autores principales: | , , , , |
| Formato: | Artículo |
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American Chemical Society
8/17/2011
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| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=hlh&AN=65264456&site=ehost-live header: @attributes: shortDbName: hlh uiTerm: 65264456 longDbName: Humanities International Complete uiTag: AN controlInfo: bkinfo: jinfo: jid: 00027863 ACS jtl: Journal of the American Chemical Society issn: 00027863 maglogo: N pubinfo: dt: 8/17/2011 vid: 133 iid: 32 pid: 997 pub: American Chemical Society artinfo: ui: 65264456 10.1021/ja201557w ppf: 12544 ppct: 12 formats: tig: atl: Time-Resolved Events on the Reaction Pathway of Transcript Initiation by a Single-Subunit RNA Polymerase: Raman Crystallographic Evidence. aug: au: Yuanyuan Chen Basu, Ritwika Gleghorn, Michael L. Murakami, Katsuhiko S. Carey, Paul R. affil: Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, United States Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, United States su: RNA polymerases Raman effect Nucleosides Phosphodiesters DNA Enzymes sug: subj: RNA polymerases Raman effect Nucleosides Phosphodiesters DNA Enzymes ab: The nucleotidyl transfer reaction leading to formation of the first phosphodiester bond has been followed in real time by Raman microscopy, as it proceeds in single crystals of the N4 phage virion RNA polymerase (RNAP). The reaction is initiated by soaking nucleoside triphosphate (NTP) substrates and divalent cations into the RNAP and promoter DNA complex crystal, where the phosphodiester bond formation is completed in about 40 min. This slow reaction allowed us to monitor the changes of the RNAP and DNA conformations as well as bindings of substrate and metal through Raman spectra taken every 5 min. Recently published snapshot X-ray crystal structures along the same reaction pathway assisted the spectroscopic assignments of changes in the enzyme and DNA, while isotopically labeled NTP substrates allowed differentiation of the Raman spectra of bases in substrates and DNA. We observed that substrates are bound at 2-7 min after soaking is commenced, the O-helix completes its conformational change, and binding of both divalent metals required for catalysis in the active site changes the conformation of the ribose triphosphate at position +1. These are followed by a slower decrease of NTP triphosphate groups due to phosphodiester bond formation that reaches completion at about 15 min and even slower complete release of the divalent metals at about 40 min. We have also shown that the O-helix movement can be driven by substrate binding only. The kinetics of the in crystallo nucleotidyl transfer reaction revealed in this study suggest that soaking the substrate and metal into the RNAP-DNA complex crystal for a few minutes generates novel and uncharacterized intermediates for future X-ray and spectroscopic analysis. pubtype: Academic Journal doctype: Article src: R language: English refInfo: copyright: @attributes: flag: Y dt: @attributes: year: 2011 holdings: @attributes: islocal: N |
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