Analysis of DNA bend structure of promoter regulatory regions of xylene-metabolizing genes on the Pseudomonas TOL plasmid.

Abstract

The transcription of both the upper operon (OP1) coding for m-xylene-degrading enzymes and the positive regulatory gene xylS on the TOL plasmid depends on sigma 54-RNA polymerase and requires the activator protein XylR that binds to the cis-acting upstream regulatory sequence of each promoter. For transcription of OP1 in Escherichia coli, integration host factor (IHF) is also required. IHF binds to DNA between the upstream regulatory sequence and the promoter sequence of OP1. We showed that IHF induced a DNA bend in the promoter regulatory region of OP1 upon its binding, supporting the DNA-loop model for the activation of OP1 transcription. In contrast to OP1, the transcriptional activation of xylS does not require IHF. In the absence of IHF, the promoter regulatory region of xylS promoter was shown to have a weak but significant intrinsic DNA bend, which may be involved in stabilizing the DNA-loop structure for the activation of xylS transcription. When IHF was highly produced in E. coli, the xylS transcription was repressed. We found two weak binding sites for IHF, each overlapping with the promoter sequence and the upstream regulatory sequence. The IHF binding to these sites might result in repression of xylS expression by overproduced IHF. Evidence is presented that binding of IHF to these sites induces a DNA bend.