Negative supercoiling induces spontaneous unwinding of a bacterial promoter

Abstract

We have examined the influence of negative supercoiling on the DNA structure of a bacterial promoter (tyrT from Escherichia coli), the transcriptional activity of which is strongly enhanced by torsional stress in vitro. Certain regions of this promoter become sensitive to digestion by single-strand-specific S1 nuclease as a consequence of negative superhelicity. These regions occur with high frequency (1 per 30-50 bp) and are normally centered on a TpA doublet. The major positions of cleavage are located in and around the -10 sequence TATGATG, the unwinding of which is a prerequisite for gene expression. An apparently trivial change in the -10 sequence from TATGATG to TATGAAG reduces both transcriptional activity and S1 nuclease sensitivity at least 10-fold. Thus the nuclease sensitivity of the promoter correlates strongly with its biological function; and both of these phenomena correlate with certain sequence-dependent structural properties of the DNA.