Transcriptional coupling between the divergent promoters of a prototypic LysR-type regulatory system, the ilvYC operon of Escherichia coli | JoVE Visualize

Transcriptional coupling between the divergent promoters of a prototypic LysR-type regulatory system, the ilvYC operon of Escherichia coli

K Y Rhee¹, M Opel, E Ito

¹Department of Microbiology, College of Medicine, University of California, Irvine, CA 92697, USA.

Proceedings of the National Academy of Sciences of the United States of America|December 10, 1999

View abstract on PubMed

Summary

Divergent transcription in the ilvYC operon of Escherichia coli affects promoter activity through DNA supercoiling. This transcriptional coupling helps coordinate gene expression with environmental conditions.

Area of Science:

Molecular Biology
Genetics
Biochemistry

Background:

The twin-domain model proposes that closely spaced, divergent promoters influence each other's transcriptional activity via transcriptionally induced negative DNA supercoiling.
This promoter arrangement is common in bacterial LysR-type-regulated operons.
The ilvYC operon in Escherichia coli serves as a model system for studying LysR-type regulation.

Purpose of the Study:

To investigate the effects of divergent transcription on the ilvYC operon in Escherichia coli.
To determine the role of DNA supercoiling in the observed transcriptional coupling between the ilvC and ilvY promoters.

Main Methods:

Utilized double-reporter constructs with lacZ and galK genes to assess in vivo transcription from the ilvC and ilvY promoters, respectively.
Performed in vitro transcription assays using various DNA topoisomers to analyze superhelicity-dependent promoter activity.
Examined the impact of novobiocin, a drug that decreases DNA superhelicity, on promoter activity.

Main Results:

A mutation in the ilvY promoter significantly reduced ilvC promoter transcription, while a mutation in the ilvC promoter had minimal effect on ilvY transcription.
In vitro studies revealed that ilvC promoter activity increased with superhelicity, whereas ilvY promoter activity showed an optimum at mid-physiological superhelicity.
Novobiocin treatment decreased negative superhelicity, leading to increased ilvY and decreased ilvC promoter activity, consistent with in vitro predictions.

Conclusions:

Transcriptional coupling between the ilvC and ilvY promoters is dependent on DNA supercoiling.
This supercoiling-mediated regulation is crucial for coordinating the basal expression of the ilvYC operon.
The coordinated expression likely adapts the operon's activity to the cell's nutritional and environmental status.