The transcription bubble of the RNA polymerase-promoter open complex exhibits conformational heterogeneity and millisecond-scale dynamics: Implications for transcription start-site selection

Nicole C. Robb, Thorben Cordes, Ling Chin Hwang, Kristofer Gryte, Diego Duchi, Timothy D. Craggs, Yusdi Santoso, Shimon Weiss, Richard H. Ebright, Achillefs N. Kapanidis

Research output: Contribution to journalArticlepeer-review

61 Scopus citations

Abstract

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 (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 RP o 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.

Original languageEnglish
Pages (from-to)875-885
Number of pages11
JournalJournal of Molecular Biology
Volume425
Issue number5
DOIs
StatePublished - 11 Mar 2013
Externally publishedYes

Bibliographical note

Funding Information:
We thank E. Fodor (Sir William Dunn School of Pathology, University of Oxford) for reagents and access to experimental facilities. T.C. was supported by a Marie Curie Intra-European Fellowship ( PIEF-GA-2009-255075 ) and Worcester College (University of Oxford) , and A.N.K. was supported by a European Commission Seventh Framework Program grant ( FP7/2007-2013 HEALTH-F4-2008-201418 ) and a Biotechnology and Biological Sciences Research Council grant ( BB/H01795X/1 ). A.N.K. and S.W. were supported by National Institutes of Health grant GM069709 . R.H.E. was supported by the National Institutes of Health grant GM41376 and a Howard Hughes Medical Investigatorship .

Keywords

  • DNA scrunching
  • RNA polymerase
  • single-molecule FRET
  • start-site selection
  • transcription initiation

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