Analysis of the binding site of the LysR-type transcriptional activator TcbR on the tcbR and tcbC divergent promoter sequences.

The TcbR transcriptional activator protein, which is encoded by the tcbR gene of Pseudomonas sp. strain P51 (J. R. van der Meer, A. C. J. Frijters, J. H. J. Leveau, R. I. L. Eggen, A. J. B. Zehnder, and W. M. de Vos, J. Bacteriol. 173:3700-3708, 1991), was purified from overproducing Escherichia coli cells by using a two-step chromatographic procedure. Subsequent use of TcbR in gel mobility shift assays with progressively shortened portions of a DNA fragment containing the divergent promoter sequences of the tcbR gene and the tcbCDEF operon showed that the direct binding site of TcbR is located between positions -85 to -40 relative to the tcbCDEF transcriptional start site, containing a LysR-type recognition sequence motif (T-N11-A). DNase I footprinting experiments revealed that TcbR protected an area on both strands of the intercistronic region which was actually larger than this binding site (from positions -74 to -24). This stretch of protected DNA was interrupted by a region (positions -52 to -37) which became strongly hypersensitive to DNase I digestion upon addition of TcbR, suggesting that TcbR induces a bend in the DNA at this site.