A recombinant Mycobacterium tuberculosis in vitro transcription system

Abstract

In vitro transcription constitutes an important tool in the study of the regulation of gene expression. Here, we present a fast and easy procedure to prepare Mycobacterium tuberculosis RNA polymerase for in vitro transcription assays. RNA polymerase is assembled from recombinant proteins expressed in Escherichia coli, thus eliminating the need for biosafety containment facilities, and is mixed with any of the 13 M. tuberculosisσ factors. We show that the recombinant RNA polymerase is free from contaminating σ factors, produces transcriptional start sites matching those characterized in vivo and allows the formal identification of σ factors involved in the expression of genes of interest. © 2005 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.