Sequence Determinants for the Recognition of the Fork Junction DNA Containing the −10 Region of Promoter DNA by E. coli RNA Polymerase†

It has been recently suggested that E. coli RNA polymerase can specifically recognize a fork junction DNA structure, suggesting a possible role for such interaction in promoter DNA melting [Guo, Y., and Gralla, J. D. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 11655−11660]. We have determined here quantitatively, using a site-specific binding assay, the effects of base substitutions within the conserved −10 hexamer in the context of a short fork junction DNA on binding to RNA polymerase. Adenine at position −11 and thymine at position −7 were found to be critical for sequence-specific recognition of the DNA. The identities of bases at positions −9 and −8 were found to be not important for the binding whereas replacement of bases at positions −12 and −10 had a mild negative effect on the binding affinity. It was found that for the binding of fork DNA to RNA polymerase, specific sequence recognition was more important than specific recognition of fork junction DNA structure. The pattern of relative importance ...