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High-throughput Purification of Affinity-tagged Recombinant Proteins
Published on: August 26, 2012
Regulated communication between the upstream face of RNA polymerase and the beta' subunit jaw domain
Siva R Wigneshweraraj1, Patricia C Burrows, Sergei Nechaev
1Department of Biological Sciences, Imperial College London, London SW7 2AZ, UK.
Abstract:
We used bacteriophage T7-encoded transcription inhibitor gene protein 2 (gp2) as a probe to study the contribution of the Escherichia coli RNA polymerase (RNAP) beta' subunit jaw domain--the site of gp2 binding--to activator and ATP hydrolysis-dependent open complex formation by the sigma(54)-RNAP. We show that, unlike sigma(70)-dependent transcription, activated transcription by sigma(54)-RNAP is resistant to gp2. In contrast, activator and ATP hydrolysis-independent transcription by sigma(54)-RNAP is highly sensitive to gp2. We provide evidence that an activator- and ATP hydrolysis-dependent conformational change involving the beta' jaw domain and promoter DNA is the basis for gp2-resistant transcription by sigma(54)-RNAP. Our results establish that accessory factors bound to the upstream face of the RNAP, communicate with the beta' jaw domain, and that such communication is subjected to regulation.
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