A recombinant Mycobacterium tuberculosis in vitro transcription system.
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. tuberculosissigma factors. We show that the recombinant RNA polymerase is free from contaminating sigma factors, produces transcriptional start sites matching those characterized in vivo and allows the formal identification of sigma factors involved in the expression of genes of interest.
Jacques
Jean-François
JF
Centre de recherche sur les mécanismes du fonctionnement cellulaire, Université de Sherbrooke, Sherbrooke, QC, Canada.
Rodrigue
Sébastien
S
Brzezinski
Ryszard
R
Gaudreau
Luc
L
D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000494:pharmacology; D012808:Sigma Factor / Q000096:biosynthesis / Q000235:genetics; D014158:Transcription, Genetic / Q000502:physiology*
D011994:Recombinant Proteins; D012808:Sigma Factor; D012321:DNA-Directed RNA Polymerases