Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 425, Issue 5, Pages 875-885Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2012.12.015
Keywords
RNA polymerase; transcription initiation; start-site selection; single-molecule FRET; DNA scrunching
Categories
Funding
- Marie Curie Intra-European Fellowship [PIEF-GA-2009-255075]
- Worcester College (University of Oxford)
- European Commission Seventh Framework [FP7/2007-2013 HEALTH-F4-2008201418]
- Biotechnology and Biological Sciences Research Council [BB/H01795/1]
- National Institutes of Health [GM069709, GM41376]
- Howard Hughes Medical Investigatorship
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [1244098] Funding Source: National Science Foundation
- Biotechnology and Biological Sciences Research Council [BB/H01795X/1] Funding Source: researchfish
- BBSRC [BB/H01795X/1] Funding Source: UKRI
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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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