Abstract

Chromatin immunoprecipitation, followed by quantification of immunoprecipitated DNA, can be used to measure RNA polymerase binding to any DNA segment in Escherichia coli. By calibrating measurements against the signal from a single RNA polymerase bound at a single promoter, we can calculate both promoter occupancy levels and the flux of transcribing RNA polymerase through transcription units. Here, we have applied the methodology to the E. coli lactose operon promoter. We confirm that promoter occupancy is limited by recruitment and that the supply of RNA polymerase to the lactose operon promoter depends on its location in the E. coli chromosome. Measurements of RNA polymerase binding to DNA segments within the lactose operon show that flux of RNA polymerase through the operon is low, with, on average, over 18 s elapsing between the passage of transcribing polymerases. Similar low levels of flux were found when semi-synthetic promoters were used to drive transcript initiation, even when the promoter elements were changed to ensure full occupancy of the promoter by RNA polymerase.This article is part of the themed issue ‘The new bacteriology’.

Highlights

  • Many bacteria rely on transcription regulation in order to adapt to fluctuating environments

  • This often involves the interaction of regulatory activator proteins at or near promoters, which results in recruitment of the DNA-dependent RNA polymerase (RNAP) and subsequent transcript initiation and gene expression

  • Few studies have addressed directly the issue of the number of RNAP molecules that engage with individual transcription units, and, to date, most calculations of RNAP flux through genes are based on estimates that work backwards from measured levels of RNA synthesis [4 – 6]

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Summary

Introduction

Many bacteria rely on transcription regulation in order to adapt to fluctuating environments. This often involves the interaction of regulatory activator proteins at or near promoters, which results in recruitment of the DNA-dependent RNA polymerase (RNAP) and subsequent transcript initiation and gene expression. We exploit the properties of the drug rifampicin, which blocks RNAP bound at promoters [9,12 – 14], to calibrate our ChIP measurements. This allows an absolute measure of promoter occupancy and RNAP flux through downstream genes. License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited

Results
OriC dif lpL
Discussion
Findings
We are grateful to the EU Framework Programme

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