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

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• 作者：Nicole C. Robb¹ ; ^&dagger ; ; Thorben Cordes¹ ; ^&dagger ; ; ¹ ; Ling Chin Hwang¹ ; ^&dagger ; ; ¹ ; Kristofer Gryte¹ ; Diego Duchi¹ ; Timothy D. Craggs¹ ; Yusdi Santoso¹ ; Shimon Weiss² ; Richard H. Ebright³ ; Achillefs N. Kapanidis¹ ; ^{a.kapanidis1@physics.ox.ac.uk}
• 关键词：RNAP ; RNA polymerase ; smFRET ; single-molecule FRET ; dsDNA ; double-stranded DNA ; BVA ; burst variance analysis
• 刊名：Journal of Molecular Biology
• 出版年：2013
• 出版时间：11 March, 2013
• 年：2013
• 卷：425
• 期：5
• 页码：875-885
• 全文大小：1005 K

文摘

Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts ~ 14 bp around the transcription start site and forms a single-stranded ¡°transcription bubble¡± within a catalytically active RNAP-DNA open complex (RP_o). 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¡ä 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 RP_o. 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 RP_o with single-molecule FRET (F?rster resonance energy transfer). We obtain experimental evidence for dynamic flexibility in RP_o using different FRET rulers and labeling positions. An analysis of FRET distributions of RP_o using burst variance analysis reveals conformational fluctuations in RP_o 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 RP_o. Our study marks the first experimental observation of conformational dynamics in the transcription bubble of RP_o and indicates that DNA dynamics within the bubble affect the search for transcription start sites.