The promoter search mechanism of E. coli RNA polymerase is dominated by three–dimensional diffusion

1] Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, USA. [2].
Nature Structural & Molecular Biology (Impact Factor: 13.31). 12/2012; 20(2). DOI: 10.1038/nsmb.2472
Source: PubMed


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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    • "transient collisions between ORC and the DNA. We estimate that at 1 nM ORC, this collision frequency should be $300 s À1 along each l ARS1 molecule (Wang et al., 2013). However, after several minutes of incubation, the number of ORC molecules per DNA is several orders of magnitude lower than if every collision resulted in stable binding (Figure 1D). "
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    • "On the other hand, E. coli RNA polymerase appeared to encounter promoters by direct collision without significant sliding [75]. This might be the ideal search mechanism in the case of high intracellular protein concentrations [75] and enforces the question whether facilitated diffusion actually plays a role for most DNA-binding proteins in vivo. "
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