4.2 Article

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

Journal

PROTEIN EXPRESSION AND PURIFICATION
Volume 69, Issue 2, Pages 235-242

Publisher

ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2009.09.022

Keywords

RNA polymerase; Sigma factors; Promoters; In vitro transcription

Funding

  1. Council of Scientific and Industrial Research
  2. Department of Biotechnology, Government of India

Ask authors/readers for more resources

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. (C) 2009 Elsevier Inc. All rights reserved.

Authors

I am an author on this paper
Click your name to claim this paper and add it to your profile.

Reviews

Primary Rating

4.2
Not enough ratings

Secondary Ratings

Novelty
-
Significance
-
Scientific rigor
-
Rate this paper

Recommended

No Data Available
No Data Available