DOI: 10.1101/494534Dec 13, 2018Paper

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

BioRxiv : the Preprint Server for Biology
Iddo HellerGijs J.L. Wuite

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 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.

Related Concepts

Diagnostic Imaging
DNA
Escherichia coli
DNA-Directed RNA Polymerase
Transcription, Genetic
T-DNA
Promoter
Surface
Three-dimensional
Analysis

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