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

Abstract

ABSTRACTThe 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 σ70 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 σ70 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 μM RNAP. The results suggest facilitated diffusion occurs in promoter search by RNAP, even at the relatively high, 0.2-0.6 μM, concentrations of RNAP in cells.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 three-dimensional 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.