Purification of RNA polymerase from mycobacteria for optimized promoter-polymerase interactions

China, Arnab and Nagaraja, Valakunja (2010) Purification of RNA polymerase from mycobacteria for optimized promoter-polymerase interactions. In: Protein Expression and Purification, 69 (2). pp. 235-242.

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Abstract

In vitro transcription analysis is important to understand the mechanism of transcription. Various assays for the analysis of initiation, elongation and termination form the basis for better understanding of the process. Purified RNA polymerase (RNAP) with high specific activity is necessary to carry out variety of these specific reactions. The RNAP purified from Mycobacterium smegmatis from exponential phase showed low promoter specificity in promoter-polymerase interaction studies. This is due to the presence of a large number of sigma factors during exponential phase and under-representation of sigma(A) required for house-keeping transcription. We describe an in vivo reconstitution of RNAP holoenzyme with sigma(A) and its purification, which resulted in holoenzyme with stoichiometric sigma(A) content. The reconstituted holoenzyme showed enhanced promoter-specific binding and promoter-specific-transcription activity compared to the enzyme isolated using standard procedure. Such in vivo reconstitution of stoichiometric holoenzyme could facilitate promoter-specific transcription assays, especially in organisms which encode a large number of sigma factors.

Item Type:	Journal Article
Publication:	Protein Expression and Purification
Publisher:	Elsevier Science
Additional Information:	copyright of this article belongs to Elsevier Science.
Keywords:	RNA polymerase;Sigma factors;Promoters;In vitro transcription.
Department/Centre:	Division of Biological Sciences > Microbiology & Cell Biology
Date Deposited:	09 Jan 2010 08:07
Last Modified:	19 Sep 2010 05:53
URI:	http://eprints.iisc.ac.in/id/eprint/25300

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