One-dimensional sliding assists σ⁷⁰-dependent promoter binding by Escherichia coli RNA polymerase

Abstract

The search for a promoter on DNA by RNA polymerase (RNAP) is an obligatory first step in transcription. The role of facilitated diffusion during promoter search has been controversial.Here, we re-assessed facilitated diffusion in promoter search by imaging motions of single molecules of Escherichia coli RNAP σ 70 holoenzyme on single DNA molecules suspended between optical traps in a manner that absolutely avoided interactions with surfaces. The assay enabled us to observe unambiguous one-dimensional sliding of RNAP  70 holoenzyme for thousands of DNA base pairs during promoter search. Analysis of binding kinetics revealed short binding events on nonspecific DNA (0.4 s), intermediate binding events on A/T-rich DNA (1.6 s), and long binding events at or near promoters (>300 s). We estimate a lower bound for the "diffusion facilitation threshold"the RNAP concentration at which three-dimensional search and one-dimensional sliding contribute equally to promoter binding of 0.2 M RNAP. The results suggest facilitated diffusion occurs in promoter search by RNAP, even at the relatively high, 0.2-0.6 M, concentrations of RNAP in cells. 3 Significance statementThe flow of genetic information from DNA to RNA is of central importance to living systems, and it can only start after an RNA polymerase (RNAP) has found a promoter site.But how does this enzyme find promoter sites on DNA in the first place? In recent years, debate on this topic has favored a promoter search mechanism that is dominated by threedimensional diffusion of RNAP, rather than by one-dimensional sliding of RNAP on DNA.Here, we designed an improved single-molecule assay that unambiguously revealed extensive one-dimensional sliding of RNAP on DNA. Our results imply that, at the RNAP concentrations in living cells, the promoter-search process is facilitated by one-dimensional sliding on DNA.

RNA polymerase clamp conformational dynamics: long-lived states and modulation by crowding, cations, and nonspecific DNA binding

“…However, although our results argue against the proposal of a mostly open clamp RNAP molecule sliding along the genomic DNA with transient clamp closures punctuating the promoter search process, the observation that some slow clamp opening-closing transitions do take place when bound to non-specific DNA, raises the possibility that clamp conformational dynamics may play a role during promoter search process. Recent studies combining single-molecule fluorescence measurements with optical tweezers have been able to observe long range 1D-sliding of RNAP molecules on doubly tethered λ DNA ( 39 ). A combination of methods used in the aforementioned study with reagents developed here should enable us to capture clamp conformations of actively diffusing RNAP molecules along DNA and provide more mechanistic detail about the role of clamp dynamics during facilitated diffusion of RNAP.…”

Quantitative parameters of bacterial RNA polymerase open-complex formation, stabilization and disruption on a consensus promoter

“…This search probably proceeds by a combination of modes, i.e. holo sliding along the DNA duplex (10)(11)(12)(13)(14)(15), holo hopping from a DNA binding site to another nearby site (16) and holo diffusing through the bulk solution (17). The holo docks on the promoter via concerted interactions with specific promoter regions known as UP (from around -60 to -40 region of the promoter relative to transcription start site at +1 position), -35, spacer, and -10 elements (8,(18)(19)(20)(21)(22).…”