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The promoter-search mechanism of Escherichia coli RNA polymerase is dominated by three-dimensional diffusion

Nature Structural & Molecular Biology volume 20, pages 174–181 (2013)Cite this article

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Abstract

Gene expression, DNA replication and genome maintenance are all initiated by proteins that must recognize specific targets from among a vast excess of nonspecific DNA. For example, to initiate transcription, Escherichia coli RNA polymerase (RNAP) must locate promoter sequences, which compose <2% of the bacterial genome. This search problem remains one of the least understood aspects of gene expression, largely owing to the transient nature of search intermediates. Here we visualize RNAP in real time as it searches for promoters, and we develop a theoretical framework for analyzing target searches at the submicroscopic scale on the basis of single-molecule target-association rates. We demonstrate that, contrary to long-held assumptions, the promoter search is dominated by three-dimensional diffusion at both the microscopic and submicroscopic scales in vitro, which has direct implications for understanding how promoters are located within physiological settings.

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Figure 1: Single-molecule DNA-curtain assay for promoter-specific binding by RNA polymerase.
Figure 2: Visualizing single molecules of RNA polymerase as they search for and engage promoters.
Figure 3: Single-molecule kinetics reveal that the promoter search is dominated by 3D diffusion.
Figure 4: Protein concentration exerts a dominant influence on target searches, even for proteins capable of sliding on DNA.
Figure 5: Increasingly complex environments encountered during in vivo searches.

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Acknowledgements

We thank M. Gottesman, R. Gonzalez and the Greene laboratory for assistance and discussion throughout this work. We thank M. Gottesman, D. Duzdevich and members of our laboratories for carefully reading the manuscript. We thank R. Landick (University of Wisconsin–Madison, Madison, Wisconsin, USA) for providing RNAP expression constructs. This work was supported by US National Institutes of Health grants GM074739 (E.C.G.) and F32GM80864 (I.J.F.) and training grant T32GM00879807 (J.G.), a US National Science Foundation Award (E.C.G.) and by the Howard Hughes Medical Institute.

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  1. Ilya J Finkelstein & Jason Gorman

    Present address: Present addresses: Department of Chemistry and Institute for Cellular & Molecular Biology, University of Texas at Austin, Austin, Texas, USA (I.J.F.) and Vaccine Research Center, National Institutes of Health, Bethesda, Maryland, USA (J.G.).,

  2. Feng Wang and Sy Redding: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, USA

    Feng Wang, Ilya J Finkelstein & Eric C Greene

  2. Department of Chemistry, Columbia University, New York, New York, USA

    Sy Redding & David R Reichman

  3. Department of Biological Sciences, Columbia University, New York, New York, USA

    Jason Gorman

  4. Howard Hughes Medical Institute, Columbia University, New York, New York, USA

    Eric C Greene

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Contributions

F.W. collected the RNAP experimental data, and F.W. and S.R. analyzed the data. S.R. developed the theoretical analysis, conducted theoretical calculations, assisted with the RNAP data collection and collected the data for the lac repressor. D.R.R. assisted with development of the theory. I.J.F. assisted in establishing the single-molecule assays for QD-RNAP and QD-lac repressor. J.G. developed the substrate for the DIG-QD measurements and collected the corresponding data. E.C.G. supervised the project, and all authors co-wrote the paper.

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Correspondence to Eric C Greene.

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Wang, F., Redding, S., Finkelstein, I. et al. The promoter-search mechanism of Escherichia coli RNA polymerase is dominated by three-dimensional diffusion. Nat Struct Mol Biol 20, 174–181 (2013). https://doi.org/10.1038/nsmb.2472

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