Abstract
Sequence-specific DNA binding proteins must quickly bind to their target sequence despite the enormously larger amount of non-target DNA present in cells. RNA polymerases (or associated general transcription factors) are hypothesized to reach promoter sequences by a facilitated diffusion (FD) mechanism. In FD, a protein first binds to non-target DNA and then reaches the target by a one-dimensional sliding search. We tested whether Escherichia coli σ54 RNA polymerase reaches a promoter by FD using a new variant of the CoSMoS multi-wavelength single-molecule fluorescence colocalization microscopy technique. The experiments directly compared the rates of initial polymerase binding to and dissociation from promoter and/or non-promoter DNAs measured in the same sample under identical conditions. Binding to a non-promoter DNA was much slower than to a promoter-containing DNA of the same length, indicating that the observed sequence non-specific binding is not on the pathway to promoter binding. Truncation of one of the DNA segments flanking the promoter to a length as short as 7 bp, or lengthening it to > 3000 bp, did not significantly alter the rate of promoter-specific binding. The rate of polymerase binding to a single promoter sequence is equivalent to that observed for binding to >3000 bp of non-promoter DNA. These results exclude FD over distances larger than ∼7 bp from playing any significant role in promoter search and also exclude other mechanisms (e.g., “hopping”) that are mediated by flanking DNA. Instead, the data support a direct binding mechanism in which σ54 RNA polymerase reaches the promoter by simple three-dimensional diffusion through solution, and they suggest that binding is accelerated by recognition of atypical structural or dynamic features of DNA within the promoter sequence.