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Expression, Autoregulation, and DNA Binding Properties of the Mycobacterium tuberculosis TrcR Response Regulator.
Shelley E. Haydel, 2002.The TrcRS two-component system of Mycobacterium tuberculosis is comprised of the TrcS histidine kinase and the TrcR response regulator, which is homologous to the OmpR class of DNA binding response regulators . Reverse transcription-PCRs with total RNA showed that the trcR and trcS two-component system genes are transcribed in broth-grown M . tuberculosis . Analysis of the trcR and trcS genes using various SCOTS (selective capture of transcribed sequences) probes also confirmed that these genes are expressed in broth-grown cultures and after 18 h of M . tuberculosis growth in cultured human primary macrophages . To determine if the TrcR response regulator is autoregulated, a trcR-lacZ fusion plasmid and a TrcR expression plasmid were cotransformed into Escherichia coli . Upon induction of the TrcR protein, there was a >500-fold increase in ß-galactosidase activity from the trcR-lacZ fusion, indicating that TrcR is involved in transcriptional autoactivation . Gel mobility shift assays with the trcR promoter and TrcR established that the response regulator was autoregulating via direct binding . By use of a delimiting series of overlapping trcR PCR fragments in gel mobility shift assays with TrcR, an AT-rich region of the trcR promoter was shown to be essential for TrcR binding . Additionally, this AT-rich sequence was protected by TrcR in DNase I protection assays . To further analyze the role of the AT-rich region in TrcR autoregulation, the trcR promoter was mutated and analyzed in lacZ transcriptional fusions in the presence of TrcR . Alteration of the AT-rich sequence in the trcR promoter resulted in the loss of trcR transcriptional activation in the presence of TrcR . This report indicates that the M . tuberculosis TrcR response regulator activates its own expression by interacting with the AT-rich sequence of the trcR promoter .

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