Optimization of a two-plasmid system for the identification of promoters recognized by RNA polymerase containing Mycobacterium tuberculosis stress response sigma factor, sigmaF
Language English Country United States Media print
Document type Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.
PubMed
15881404
DOI
10.1007/bf02931550
Knihovny.cz E-resources
- MeSH
- Bacterial Proteins genetics metabolism MeSH
- DNA-Directed RNA Polymerases genetics metabolism MeSH
- Escherichia coli genetics metabolism MeSH
- Cloning, Molecular MeSH
- Mycobacterium tuberculosis genetics metabolism MeSH
- Plasmids genetics MeSH
- Promoter Regions, Genetic * MeSH
- Amino Acid Sequence MeSH
- Base Sequence MeSH
- Sequence Analysis, DNA MeSH
- Sigma Factor genetics metabolism MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
- Names of Substances
- Bacterial Proteins MeSH
- DNA-Directed RNA Polymerases MeSH
- FliA protein, Bacteria MeSH Browser
- Sigma Factor MeSH
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.
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