Abstract
Bacterial RNA polymerases (RNAPs) have two flexibly tethered alpha C-terminal domains that bind DNA. Interaction between CTDs and some promoter sequences is known to accelerate transcription initiation, but the mechanism is unclear. Here, we used single-molecule multiwavelength fluorescence microscopy to test how the sequence non-specific binding kinetics of RNAP to DNA change as compared to mutants that lack one or both αCTDs. We find that even though the CTD is small compared to the whole RNAP, the presence of the two CTDs accelerates DNA binding by roughly 35-fold; 10-fold if one CTD is present. In contrast, the presence of αCTDs did not have a measurable effect on RNAP-DNA complex lifetimes. We explain how αCTDs dramatically accelerate RNAP binding to DNA using a three-state model that assumes a transient intermediate state of binding where only the αCTD(s) are bound to DNA, tethering the non-CTD RNAP in the vicinity of DNA. Assumed model parameters are validated using Brownian dynamics simulations of the RNAP-DNA association reaction. The combination of the single-molecule experiments and theory suggests that adding a flexible DNA-binding tether is a general mechanism to substantially accelerate the diffusion-limited binding of a large protein to DNA.