Evidence for a Tyrosine-Adenine Stacking Interaction and for a Short-lived Open Intermediate Subsequent to Initial Binding of Escherichia coli RNA Polymerase to Promoter DNA

JOURNAL OF MOLECULAR BIOLOGY

卷:385

Article

Schroeder, Lisa A.¹  Gries, Theodore J.²  Saecker, Ruth M.²  Record, M. Thomas, Jr.²  Harris, Michael E.¹  deHaseth, Pieter L.^1,3 

[1] Case Western Reserve Univ, Ctr RNA Mol Biol, Cleveland, OH 44106 USA

[2] Univ Wisconsin, Dept Biochem, Madison, WI 53706 USA

[3] Case Western Reserve Univ, Dept Biochem, Cleveland, OH 44106 USA

关键词: RNA polymerase;    sigma;    strand opening;    stacking;    open intermediate;   

DOI  :  10.1016/j.jmb.2008.10.023

来源: Elsevier

【 摘 要 】

Bacterial RNA polymerase and a sigma transcription factor form an initiation-competent open complex at a promoter by disruption of about 14 base pairs. Strand separation is likely initiated at the highly conserved -11 A-T base pair. Amino acids in conserved region 2.3 of the main Escherichia coli sigma factor, sigma(70), are involved in this process, but their roles are unclear. To monitor the fates of particular bases upon addition of RNA polymerase, promoters bearing single substitutions of the fluorescent A-analog 2-aminopurine (2-AP) at -11 and two other positions in promoter DNA were examined. Evidence was obtained for an open intermediate on the pathway to open complex formation, in which these 2-APs are no longer stacked onto their neighboring bases. The tyrosine at residue 430 in region 2.3 of sigma(70) was shown to be involved in quenching the fluorescence of a 2-AP substituted at -11, presumably through a stacking interaction. These data refine the structural model for open complex formation and reveal a novel interaction involved in DNA melting by RNA polymerase. (C) 2008 Elsevier Ltd. All rights reserved.

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