Passage of RNA polymerase from open complex to elongation mode at the Escherichia coli lacUV5 promoter: nucleolytic hypersensitivity as a probe for complex conformational changes.

In transcriptionally active complexes between RNA polymerase and promoters, the center of the melted region is hyperreactive to the nucleolytic activity of the cuprous complex of 1,10-phenanthroline (OP-Cu). In the first part of this work, using synthetic oligonucleotides and exploiting gel retardation assays, I demonstrate that DNA unpairing is not the only determinant of this hyperreactivity. Polymerase binding is directly implicated, presumably participating in the stabilization of an intermediate required for the cutting. In the second part of the work, I show that, from fine analysis of the nucleolytic pattern of lacUV5 promoter DNA towards OP-Cu and Phe OP-Cu, it is possible to locate polymerase and to characterize its contacts at any time during the early stages of transcription. This analysis provides a description of the passage from the "open complex" to the elongation mode in terms of, first, release of the upstream contacts, and second, loss of sigma subunit. Occupancy of the overlapping promoter, P2, has a positive effect on the escape of polymerase from abortive cycling. The involvement of sigma and beta subunits in the reactivity pattern is discussed with respect to previous cross-linking studies.