title: RNA polymerase approaches its promoter without long-range sliding along DNA.
abstract: Sequence-specific DNA binding proteins must quickly bind target sequences, despite the enormously larger amount of nontarget DNA present in cells. RNA polymerases (or associated general transcription factors) are hypothesized to reach promoter sequences by facilitated diffusion (FD). In FD, a protein first binds to nontarget DNA and then reaches the target by a 1D sliding search. We tested whether Escherichia coli σ(54)RNA polymerase reaches a promoter by FD using the colocalization single-molecule spectroscopy (CoSMoS) multiwavelength fluorescence microscopy technique. Experiments directly compared the rates of initial polymerase binding to and dissociation from promoter and nonpromoter DNAs measured in the same sample under identical conditions. Binding to a nonpromoter DNA was much slower than binding to a promoter-containing DNA of the same length, indicating that the detected nonspecific binding events are not on the pathway to promoter binding. Truncating one of the DNA segments flanking the promoter to a length as short as 7 bp or lengthening it to ~3,000 bp did not alter the promoter-specific binding rate. These results exclude FD over distances corresponding to the length of the promoter or longer from playing any significant role in accelerating promoter search. Instead, the data support a direct binding mechanism, in which σ(54)RNA polymerase reaches the local vicinity of promoters by 3D diffusion through solution, and suggest that binding may be accelerated by atypical structural or dynamic features of promoter DNA. Direct binding explains how polymerase can quickly reach a promoter, despite occupancy of promoter-flanking DNA by bound proteins that would impede FD.
authors.affiliation: Department of Biochemistry, Brandeis University, Waltham, MA 02454, USA.
authors.forename: Larry J
authors.initials: LJ
authors.lastname: Friedman
authors.forename: Jeffrey P
authors.initials: JP
authors.lastname: Mumm
authors.forename: Jeff
authors.initials: J
authors.lastname: Gelles
mesh_terms: D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004058:Diffusion; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051877:RNA Polymerase Sigma 54 / Q000235:genetics / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C497751:rpoN protein, E coli; D051877:RNA Polymerase Sigma 54
keywords: 1D diffusion; total internal reflection fluorescence; transcription initiation