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 su ch interaction in promoter DNA melting [Guo, Y., and Gralla, J. D, (1998) P roc, Natl. Acad. Sci. U.S.A, 95, 11655-11660]. We have determined here quan titatively, using a site-specific binding assay, the effects of base substi tutions within the conserved -10 hexamer in the context of a short fork jun ction 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 b e 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 reco gnition was more important than specific recognition of fork junction DNA s tructure. The pattern of relative importance of bases in the -10 region for binding RNA polymerase was generally consistent with the sequence conserva tion pattern observed in nature where positions -11 and -7 are the most con served. Binding experiments with a series of adenine analogues at position -11 revealed that the N1 nitrogen of adenine was a critical determinant for the preference of the adenine at this position, suggesting a mechanism for the nucleation of promoter DNA melting initiation in which RNA polymerase destabilizes duplex DNA by directly competing with the thymine of the A-T b ase pair for hydrogen bonding to the N1 position of the -11 nontemplate str and adenine.