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

• Published in: Journal of molecular biology 2013-03, Vol.425 (5), p.875-885
• Main Authors: Robb, Nicole C., Cordes, Thorben, Hwang, Ling Chin, Gryte, Kristofer, Duchi, Diego, Craggs, Timothy D., Santoso, Yusdi, Weiss, Shimon, Ebright, Richard H., Kapanidis, Achillefs N.
• Format: Article
• Language: English
• Subjects: analysis of variance; Base Sequence; Catalytic Domain; DNA; DNA - chemistry; DNA - genetics; DNA - metabolism; DNA scrunching; DNA-directed RNA polymerase; DNA-Directed RNA Polymerases - chemistry; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; energy transfer; Fluorescence Resonance Energy Transfer; genes; Humans; messenger RNA; Molecular Sequence Data; mutation; Nucleic Acid Conformation; nucleotides; promoter regions; Promoter Regions, Genetic - genetics; RNA polymerase; Sequence Homology, Nucleic Acid; single-molecule FRET; start-site selection; transcription (genetics); transcription initiation; Transcription Initiation Site; Transcription, Genetic
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2012.12.015

Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts ~14bp 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 (Förster 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. [Display omitted] ► Heterogeneity in transcription start sites is widespread and important for gene regulation. ► DNA flexibility has been suggested to be important for start-site heterogeneity. ► We tested for DNA flexibility in transcription open complexes (RPo) by single-molecule FRET. ► We provide evidence for dynamic flexibility in RPo in the millisecond timescale. ► We reprogrammed start sites by repositioning DNA versus the RNAP active site in RP.