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.

Large enzymes that must associate with DNA to perform their biological functions are expected to bind DNA only slowly because of slow enzyme diffusion. However, some DNA-binding enzymes have one or more additional small DNA-binding domains that can diffuse rapidly but are attached to the enzyme through an unstructured flexible tether. Combining single-molecule experiments, theory, and computation, we present evidence for a general mechanism by which such tethered domains can accelerate enzyme binding to DNA by orders of magnitude, while not significantly changing the duration of the DNA-bound state. We demonstrate this effect for the flexibly-tethered α-subunit C-terminal domain of bacterial RNA polymerase. The mechanism may allow the polymerase to rapidly bind DNA while minimizing non-functional sequestration on the genome.