Bacterial RNA polymerase exemplifies a general physical mechanism for accelerating protein-DNA association

Bacterial RNA polymerases (RNAPs) have two flexibly tethered α subunit C-terminal domains (α-CTDs) that bind DNA. Interaction between α-CTDs and some promoter DNA motifs is known to accelerate transcription initiation, but the physical mechanism by which it does so is unclear. We used single-molecule multiwavelength fluorescence microscopy to test how the diffusion-limited binding kinetics of core RNAP to non-promoter DNA differ from those of mutant RNAPs that lack one or both α-CTDs. We find that even though α-CTDs and their tethers are small compared to the complete RNAP molecule, the presence of two α-CTDs accelerates DNA binding by ∼10-fold and ∼55-fold respectively relative to RNAP constructs in which one or both α-CTDs are deleted. In contrast, the presence of α-CTDs did not have a detectable effect on RNAP-DNA complex lifetimes in the absence of RNA synthesis. We explain how α-CTDs achieve the dramatic acceleration of RNAP binding to DNA using a quantitative three-state kinetic model that includes a transient binding intermediate where only the α-CTD(s) are bound to DNA, tethering the rest of the RNAP in the vicinity of DNA. The model and assumed parameters are validated using Brownian dynamics simulations of the DNA association reactions for two-, one-, or zero-CTD RNAP constructs. The combination of single-molecule experiments, mathematical theory, and simulations suggests that adding a flexible DNA-binding tether is a general physical mechanism which can accelerate the diffusion-limited binding of a large protein like RNAP to DNA and quantitatively defines the conditions under which this acceleration can occur.