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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    ABSTRACT: Eukaryotic replication initiation is highly regulated and dynamic. It begins with the origin recognition complex (ORC) binding DNA sites called origins of replication. ORC, together with Cdc6 and Cdt1, mediate pre-replicative complex (pre-RC) assembly by loading a double hexamer of Mcm2-7: the core of the replicative helicase. Here, we use single-molecule imaging to directly visualize Saccharomyces cerevisiae pre-RC assembly and replisome firing in real time. We show that ORC can locate and stably bind origins within large tracts of non-origin DNA and that Cdc6 drives ordered pre-RC assembly. We further show that the dynamics of the ORC-Cdc6 interaction dictate Mcm2-7 loading specificity and that Mcm2-7 double hexamers form preferentially at a native origin sequence. Finally, we demonstrate that single Mcm2-7 hexamers propagate bidirectionally, monotonically, and processively as constituents of active replisomes. Copyright © 2015 Elsevier Inc. All rights reserved.
    Molecular Cell 04/2015; 58(3). DOI:10.1016/j.molcel.2015.03.017 · 14.02 Impact Factor
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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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    DNA repair 08/2014; 20(100). DOI:10.1016/j.dnarep.2014.02.015 · 3.11 Impact Factor
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    • "bearing binding sites for each of the roadblocks (Figure S1). We have previously shown site-specific DNA-binding on DNA curtains for QD-tagged EcoRI E111Q , LacI, FtsKabg D1121A , and RNAP (Finkelstein et al., 2010; Lee et al., 2012; Wang et al., 2013), and we verified that Tus, XerD, and XerCD were also correctly targeted to each of their cognate sites (Figure S2). "
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    ABSTRACT: In physiological settings, DNA translocases will encounter DNA-bound proteins, which must be dislodged or bypassed to allow continued translocation. FtsK is a bacterial translocase that promotes chromosome dimer resolution and decatenation by activating XerCD-dif recombination. To better understand how translocases act in crowded environments, we used single-molecule imaging to visualize FtsK in real time as it collided with other proteins. We show that FtsK can push, evict, and even bypass DNA-bound proteins. The primary factor dictating the outcome of collisions was the relative affinity of the proteins for their specific binding sites. Importantly, protein-protein interactions between FtsK and XerD help prevent removal of XerCD from DNA by promoting rapid reversal of FtsK. Finally, we demonstrate that RecBCD always overwhelms FtsK when these two motor proteins collide while traveling along the same DNA molecule, indicating that RecBCD is capable of exerting a much greater force than FtsK when translocating along DNA.
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