Optimization of a two-plasmid system for the identification of promoters recognized by RNA polymerase containing Mycobacterium tuberculosis stress response sigma factor, sigmaF.
The previously established two-plasmid system in Escherichia coli for the identification of Mycobacterium tuberculosis promoters that are recognized by RNA polymerase containing the stress response sigma factor sigmaF was optimized. Expression of the M. tuberculosis sigmaF encoded by sigF gene was under the control of the isopropyl beta-D-thiogalactopyranoside (IPTG)-dependent Ptrc promoter. A low level of IPTG induced a nontoxic but sufficient level of sigmaF to interact with the core enzyme of RNA polymerase. Such an RNA polymerase holoenzyme recognized the known sigmaF-dependent promoter, usfXp1, which was cloned in the compatible promoter probe plasmid, upstream of a promoterless lacZalpha reporter gene. Primer extension analysis of the usfXp1 promoter in the E. coli two-plasmid system after IPTG-induced expression of M. tuberculosis sigF revealed a transcription start point that was identical as in M. tuberculosis. This new system has been shown to be useful for identification of M. tuberculosis sigmaF-dependent promoters.
Homerová
D
D
Institute of Molecular Biology, Center of Excellence for Molecular Medicine, Slovak Academy of Sciences, 845 51 Bratislava, Slovakia.
Surdová
K
K
Kormanec
J
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; C064971:FliA protein, Bacteria; D012808:Sigma Factor; D012321:DNA-Directed RNA Polymerases