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


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.


  1. 1

    Haugen, S.P., Ross, W. & Gourse, R.L. Advances in bacterial promoter recognition and its control by factors that do not bind DNA. Nat. Rev. Microbiol. 6, 507–519 (2008).

    CAS  Article  Google Scholar 

  2. 2

    Browning, D.F. & Busby, S.J. The regulation of bacterial transcription initiation. Nat. Rev. Microbiol. 2, 57–65 (2004).

    CAS  Article  Google Scholar 

  3. 3

    Saecker, R.M., Record, M.T. & Dehaseth, P.L. Mechanism of bacterial transcription initiation: RNA polymerase—promoter binding, isomerization to initiation-competent open complexes, and initiation of RNA synthesis. J. Mol. Biol. 412, 754–771 (2011).

    CAS  Article  Google Scholar 

  4. 4

    Nudler, E. RNA polymerase active center: the molecular engine of transcription. Annu. Rev. Biochem. 78, 335–361 (2009).

    CAS  Article  Google Scholar 

  5. 5

    Mendoza-Vargas, A. et al. Genome-wide identification of transcription start sites, promoters and transcription factor binding sites in E. coli. PLoS ONE 4, e7526 (2009).

    Article  Google Scholar 

  6. 6

    Cho, B.K. et al. The transcription unit architecture of the Escherichia coli genome. Nat. Biotechnol. 27, 1043–1049 (2009).

    CAS  Article  Google Scholar 

  7. 7

    von Hippel, P.H. & Berg, O.G. Facilitated target location in biological systems. J. Biol. Chem. 264, 675–678 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Gorman, J. & Greene, E.C. Visualizing one-dimensional diffusion of proteins along DNA. Nat. Struct. Mol. Biol. 15, 768–774 (2008).

    CAS  Article  Google Scholar 

  9. 9

    Berg, O.G. & Blomberg, C. Association kinetics with coupled diffusional flows. Special application to the lac repressor–operator system. Biophys. Chem. 4, 367–381 (1976).

    CAS  Article  Google Scholar 

  10. 10

    Mirny, L. et al. How a protein searches for its site on DNA: the mechanism of facilitated diffusion. J. Phys. A 42, 434013 (2009).

    Article  Google Scholar 

  11. 11

    Halford, S.E. & Marko, J.F. How do site-specific DNA-binding proteins find their targets? Nucleic Acids Res. 32, 3040–3052 (2004).

    CAS  Article  Google Scholar 

  12. 12

    Berg, O.G., Winter, R.B. & von Hippel, P.H. Diffusion-driven mechanisms of protein translocation on nucleic acids. 1. Models and theory. Biochemistry 20, 6929–6948 (1981).

    CAS  Article  Google Scholar 

  13. 13

    Riggs, A.D., Bourgeois, S. & Cohn, M. The lac repressor-operator interaction. 3. Kinetic studies. J. Mol. Biol. 53, 401–417 (1970).

    CAS  Article  Google Scholar 

  14. 14

    Halford, S.E. An end to 40 years of mistakes in DNA-protein association kinetics? Biochem. Soc. Trans. 37, 343–348 (2009).

    CAS  Article  Google Scholar 

  15. 15

    Li, G.-W., Berg, O.G. & Elf, J. Effects of macromolecular crowding and DNA looping on gene regulation kinetics. Nat. Phys. 5, 294–297 (2009).

    CAS  Article  Google Scholar 

  16. 16

    Hu, T., Grosberg, A.Y. & Shklovskii, B.I. How proteins search for their specific sites on DNA: the role of DNA conformation. Biophys. J. 90, 2731–2744 (2006).

    CAS  Article  Google Scholar 

  17. 17

    Bauer, M. & Metzler, R. Generalized faciliated diffusion model for DNA-binding proteins with search and recognition states. Biophys. J. 102, 2321–2330 (2012).

    CAS  Article  Google Scholar 

  18. 18

    Kolesov, G., Wunderlich, Z., Laikova, O.N., Gelfand, M.S. & Mirny, L.A. How gene order is influenced by the biophysics of transcription regulation. Proc. Natl. Acad. Sci. USA 104, 13948–13953 (2007).

    CAS  Article  Google Scholar 

  19. 19

    Wunderlich, Z. & Mirny, L.A. Spatial effects on the speed and reliability of protein-DNA search. Nucleic Acids Res. 36, 3570–3578 (2008).

    CAS  Article  Google Scholar 

  20. 20

    Das, R.K. & Kolomeisky, A.B. Facilitated search of proteins on DNA: correlations are important. Phys. Chem. Chem. Phys. 12, 2999–3004 (2010).

    CAS  Article  Google Scholar 

  21. 21

    Singer, P. & Wu, C.W. Promoter search by Escherichia coli RNA polymerase on a circular DNA template. J. Biol. Chem. 262, 14178–14189 (1987).

    CAS  PubMed  Google Scholar 

  22. 22

    Ricchetti, M., Metzger, W. & Heumann, H. One-dimensional diffusion of Escherichia coli DNA-dependent RNA polymerase: a mechanism to facilitate promoter location. Proc. Natl. Acad. Sci. USA 85, 4610–4614 (1988).

    CAS  Article  Google Scholar 

  23. 23

    Kabata, H. et al. Visualization of single molecules of RNA polymerase sliding along DNA. Science 262, 1561–1563 (1993).

    CAS  Article  Google Scholar 

  24. 24

    Guthold, M. et al. Direct observation of one-dimensional diffusion and transcription by Escherichia coli RNA polymerase. Biophys. J. 77, 2284–2294 (1999).

    CAS  Article  Google Scholar 

  25. 25

    Harada, Y. et al. Single-molecule imaging of RNA polymerase-DNA interactions in real time. Biophys. J. 76, 709–715 (1999).

    CAS  Article  Google Scholar 

  26. 26

    Berg, J., Tymoczko, J. & Stryer, L. Biochemistry (W.H. Freeman, 2007).

  27. 27

    Roe, J.H., Burgess, R.R. & Record, M.T. Jr. Kinetics and mechanism of the interaction of Escherichia coli RNA polymerase with the lambda PR promoter. J. Mol. Biol. 176, 495–522 (1984).

    CAS  Article  Google Scholar 

  28. 28

    Friedman, L.J. & Gelles, J. Mechanism of transcription initiation at an activator-dependent promoter defined by single-molecule observation. Cell 148, 679–689 (2012).

    CAS  Article  Google Scholar 

  29. 29

    deHaseth, P.L., Zupancic, M. & Record, M.T. Jr. RNA polymerase-promoter interactions: the comings and goings of RNA polymerase. J. Bacteriol. 180, 3019–3025 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Herbert, K.M., Greenleaf, W.J. & Block, S.M. Single-molecule studies of RNA polymerase: motoring along. Annu. Rev. Biochem. 77, 149–176 (2008).

    CAS  Article  Google Scholar 

  31. 31

    Gorman, J., Fazio, T., Wang, F., Wind, S. & Greene, E.C. Nanofabricated racks of aligned and anchored DNA substrates for single-molecule imaging. Langmuir 26, 1372–1379 (2010).

    CAS  Article  Google Scholar 

  32. 32

    Gorman, J., Plys, A.J., Visnapuu, M.L., Alani, E. & Greene, E.C. Visualizing one-dimensional diffusion of eukaryotic DNA repair factors along a chromatin lattice. Nat. Struct. Mol. Biol. 17, 932–938 (2010).

    CAS  Article  Google Scholar 

  33. 33

    Gorman, J. et al. Single-molecule imaging reveals target search mechanisms during mismatch repair. Proc. Natl. Acad. Sci. USA 109, E3074–E3083 (2012).

    CAS  Article  Google Scholar 

  34. 34

    Finkelstein, I.J., Visnapuu, M.L. & Greene, E.C. Single-molecule imaging reveals mechanisms of protein disruption by a DNA translocase. Nature 468, 983–987 (2010).

    CAS  Article  Google Scholar 

  35. 35

    Simons, R.W., Hoopes, B.C., McClure, W.R. & Kleckner, N. Three promoters near the termini of IS10: pIN, pOUT, and pIII. Cell 34, 673–682 (1983).

    CAS  Article  Google Scholar 

  36. 36

    McClure, W.R. Rate-limiting steps in RNA chain initiation. Proc. Natl. Acad. Sci. USA 77, 5634–5638 (1980).

    CAS  Article  Google Scholar 

  37. 37

    Hawley, D.K. & McClure, W.R. In vitro comparison of initiation properties of bacteriophage lambda wild-type PR and x3 mutant promoters. Proc. Natl. Acad. Sci. USA 77, 6381–6385 (1980).

    CAS  Article  Google Scholar 

  38. 38

    Dayton, C.J., Prosen, D.E., Parker, K.L. & Cech, C.L. Kinetic measurements of Escherichia coli RNA polymerase association with bacteriophage T7 early promoters. J. Biol. Chem. 259, 1616–1621 (1984).

    CAS  PubMed  Google Scholar 

  39. 39

    Brunner, M. & Bujard, H. Promoter recognition and promoter strength in the Escherichia coli system. EMBO J. 6, 3139–3144 (1987).

    CAS  Article  Google Scholar 

  40. 40

    Wang, Y.M., Austin, R.H. & Cox, E.C. Single molecule measurements of repressor protein 1D diffusion on DNA. Phys. Rev. Lett. 97, 048302 (2006).

    CAS  Article  Google Scholar 

  41. 41

    Elf, J., Li, G.W. & Xie, X.S. Probing transcription factor dynamics at the single-molecule level in a living cell. Science 316, 1191–1194 (2007).

    CAS  Article  Google Scholar 

  42. 42

    Kim, J.H. & Larson, R.G. Single-molecule analysis of 1D diffusion and transcription elongation of T7 RNA polymerase along individual stretched DNA molecules. Nucleic Acids Res. 35, 3848–3858 (2007).

    CAS  Article  Google Scholar 

  43. 43

    Tafvizi, A., Huang, F., Fersht, A.R., Mirny, L.A. & van Oijen, A.M. A single-molecule characterization of p53 search on DNA. Proc. Natl. Acad. Sci. USA 108, 563–568 (2011).

    CAS  Article  Google Scholar 

  44. 44

    Berg, O.G. Orientation constraints in diffusion-limited macromolecular association. The role of surface diffusion as a rate-enhancing mechanism. Biophys. J. 47, 1–14 (1985).

    CAS  Article  Google Scholar 

  45. 45

    Austin, R.H., Karohl, J. & Jovin, T.M. Rotational diffusion of Escherichia coli RNA polymerase free and bound to deoxyribonucleic acid in nonspecific complexes. Biochemistry 22, 3082–3090 (1983).

    CAS  Article  Google Scholar 

  46. 46

    Gorman, J. et al. Dynamic basis for one-dimensional DNA scanning by the mismatch repair complex Msh2-Msh6. Mol. Cell 28, 359–370 (2007).

    CAS  Article  Google Scholar 

  47. 47

    Berg, O.G. & von Hippel, P.H. Diffusion-controlled macromolecular interactions. Annu. Rev. Biophys. Biophys. Chem. 14, 131–160 (1985).

    CAS  Article  Google Scholar 

  48. 48

    Moran, U., Philips, R. & Milo, R. Snapshot: key numbers in biology. Cell 141, 1262–1262.e1 (2010).

    Article  Google Scholar 

  49. 49

    Minton, A.P. The influence of macromolecular crowding and macromolecular confinement on biochemical reactions in physiological media. J. Biol. Chem. 276, 10577–10580 (2001).

    CAS  Article  Google Scholar 

  50. 50

    Graham, J.S., Johnson, R.C. & Marko, J.F. Concentration-dependent exchange accelerates turnover of proteins bound to double-stranded DNA. Nucleic Acids Res. 39, 2249–2259 (2011).

    CAS  Article  Google Scholar 

  51. 51

    Ishihama, A. Functional modulation of Escherichia coli RNA polymerase. Annu. Rev. Microbiol. 54, 499–518 (2000).

    CAS  Article  Google Scholar 

  52. 52

    Hammar, P. et al. The lac repressor displays faciliated diffusion in living cells. Science 336, 1595–1598 (2012).

    CAS  Article  Google Scholar 

  53. 53

    McClure, W.R. Mechanism and control of transcription initiation in prokaryotes. Annu. Rev. Biochem. 54, 171–204 (1985).

    CAS  Article  Google Scholar 

  54. 54

    So, L.H. et al. General properties of transcriptional time series in Escherichia coli. Nat. Genet. 43, 554–560 (2011).

    CAS  Article  Google Scholar 

  55. 55

    Reppas, N.B., Wade, J.T., Church, G.M. & Struhl, K. The transition between trancriptional initiation and elongation in E. coli is highly variable and often rate limiting. Mol. Cell 24, 747–757 (2006).

    CAS  Article  Google Scholar 

  56. 56

    Shaevitz, J.W., Abbondanzieri, E.A., Landick, R. & Block, S.M. Backtracking by single RNA polymerase molecules observed at near-base-pair resolution. Nature 426, 684–687 (2003).

    CAS  Article  Google Scholar 

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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.

Author information




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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The authors declare no competing financial interests.

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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).

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