New Start Site for Escherichia coli RNA Polymerase at an Engineered Short Region of Non-complementarity in Double-stranded DNA

Abstract

We have constructed versions of the bacteriophage P RM promoter containing short (9 or 12 base pairs) regions of DNA mismatches ("bubbles") which include the authentic transcription start site of the unmodified promoter. These constructs direct transcription initiation at positions near the genuine P RM start site. In addition a new start site (designated P bub) is observed in the region of non-complementarity, from which RNA synthesis proceeds in the opposite direction. The ability to initiate the divergent transcripts is specific to holo enzyme. Mapping of the P bub start sites shows that they are but a few base pairs upstream of the edge of the bubble. Thus, with respect to the single-stranded region, the location of the start site is no different for P bub than it is for open complexes at promoters. Compared with an unmodified P RM promoter, the region protected by RNA polymerase from digestion by DNase I is extended in the downstream direction (with respect to the P RM start) at the promoters bearing mismatches; this is consistent with the binding of the divergently transcribing RNA polymerase. Interestingly, cI protein represses rather than activates RNA synthesis originating in the P RM direction, indicating yet another aspect in which the complexes formed at these constructs differ from open complexes at the unmodified promoter