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 of bases in the -10 region for binding RNA polymerase was generally consistent with the sequence conservation pattern observed in nature where positions -11 and -7 are the most conserved. 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 base pair for hydrogen bonding to the N1 position of the -11 nontemplate strand adenine.