The Transcription Bubble of the RNA Polymerase-Promoter Open Complex Exhibits Conformational Heterogeneity and Millisecond-Scale Dynamics: Implications for Transcription Start-Site Selection

JOURNAL OF MOLECULAR BIOLOGY

卷:425

Article

Robb, Nicole C.¹  Cordes, Thorben¹  Hwang, Ling Chin¹  Gryte, Kristofer¹  Duchi, Diego¹  Craggs, Timothy D.¹  Santoso, Yusdi¹  Weiss, Shimon²  Ebright, Richard H.^3,4  Kapanidis, Achillefs N.¹ 

[1] Univ Oxford, Clarendon Lab, Dept Phys, Biol Phys Res Grp, Oxford OX1 3PU, England

[2] Univ Calif Los Angeles, Dept Chem & Biochem, Los Angeles, CA 90095 USA

[3] Rutgers State Univ, Dept Chem, Howard Hughes Med Inst, Piscataway, NJ 08854 USA

[4] Rutgers State Univ, Waksman Inst, Piscataway, NJ 08854 USA

关键词: RNA polymerase;    transcription initiation;    start-site selection;    single-molecule FRET;    DNA scrunching;   

DOI  :  10.1016/j.jmb.2012.12.015

来源: Elsevier

【 摘 要 】

Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts similar to 14 bp around the transcription start site and forms a single-stranded transcription bubble within a catalytically active RNAP-DNA open complex (RPo). There is significant flexibility in the transcription start site, which causes variable spacing between the promoter elements and the start site; this in turn causes differences in the length and sequence at the 5' end of RNA transcripts and can be important for gene regulation. The start-site variability also implies the presence of some flexibility in the positioning of the DNA relative to the RNAP active site in RPo,. The flexibility may occur in the positioning of the transcription bubble prior to RNA synthesis and may reflect bubble expansion (scrunching) or bubble contraction (unscrunching). Here, we assess the presence of dynamic flexibility in RPo, with single-molecule FRET (Forster resonance energy transfer). We obtain experimental evidence for dynamic flexibility in RPo, using different FRET rulers and labeling positions. An analysis of FRET distributions of RPo, using burst variance analysis reveals conformational fluctuations in RPo, in the millisecond timescale. Further experiments using subsets of nucleotides and DNA mutations allowed us to reprogram the transcription start sites, in a way that can be described by repositioning of the single-stranded transcription bubble relative to the RNAP active site within RPo,. Our study marks the first experimental observation of conformational dynamics in the transcription bubble of RPo, and indicates that DNA dynamics within the bubble affect the search for transcription start sites. (C) 2013 Elsevier Ltd. All rights reserved.

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