RNA polymerase supply and flux through the lac operon in Escherichia coli

Research output: Contribution to journalArticlepeer-review

Authors

Colleges, School and Institutes

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.

Details

Original languageEnglish
JournalRoyal Society of London. Proceedings B. Biological Sciences
Volume371
Issue number1707
Early online date26 Sep 2016
Publication statusE-pub ahead of print - 26 Sep 2016