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.1D diffusion | total internal reflection fluorescence | transcription initiation T he binding of a multisubunit RNA polymerase (RNAP) or general transcription factors to a specialized transcription promoter DNA sequence is an essential step in initiating DNA transcription in all organisms (1, 2). Control of this promoter binding step is a key mechanism by which gene expression is regulated (3). To understand how this regulation occurs, it is necessary to understand the process by which the transcription machinery efficiently finds and binds to target sequences embedded in chromosomal DNA, where targets are outnumbered by orders of magnitude excess nontarget DNA.Bacterial transcription is a convenient model system for studying promoter search, because the process can be efficiently reconstituted in vitro from purified components and bacterial RNAP holoenzymes recognize and bind directly to specific promoter DNA sequence motifs (4, 5). For the well-studied Escherichia coli RNAP, the rate at which the enzyme finds its cognate promoters can approach or even possibly exceed limits calculated for simple 3D diffusion of the protein up to the target sequence followed by direct promoter binding (6-8). Although there are uncertainties in calculating the magnitude of this limit because of uncertain contributions from orientation constraints and electr...

The site‐specific recombination pathway by which the bacteriophage λ chromosome is excised from its Escherichia coli host chromosome is a tightly regulated, highly directional, multistep reaction that is executed by a series of multiprotein complexes. Until now, it has been difficult to study the individual steps of such reactions in the context of the entire pathway. Using single‐molecule light microscopy, we have examined this process from start to finish. Stable bent‐DNA complexes containing integrase and the accessory proteins IHF (integration host factor) and Xis form rapidly on attL and attR recombination partners, and synapsis of partner complexes follows rapidly after their formation. Integrase‐mediated DNA cleavage before or immediately after synapsis is required to stabilize the synaptic assemblies. Those complexes that synapsed (∼50% of the total) yield recombinant product with a remarkable ∼100% efficiency. The rate‐limiting step of excision occurs after synapsis, but closely precedes or is concomitant with the appearance of a stable Holliday junction. Our kinetic analysis shows that directionality of this recombination reaction is conferred by the irreversibility of multiple reaction steps.

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