Major Groove Recognition Elements in the Middle of the T7 RNA Polymerase Promoter

Abstract

T7 RNA polymerase recognizes a relatively small promoter extending only 17 base pairs upstream from the start site for transcription. A model for this recognition suggests that the enzyme interacts with the major groove of duplex DNA in the region centered at position -9 [Muller, D.K., et al. (1989) Biochemistry 28, 3306-3313], and recent kinetic analyses of promoters containing base analogs at positions -10 and -11 have provided support for this model [Schick, C., & Martin, C.T. (1993) Biochemistry 32, 4275-4280; Schick, C., & Martin, C.T. (1995) Biochemistry 34, 666-672]. In the current work, we extend this analysis across the proposed major groove, identifying specific base functional group contacts at positions -9 through -5. Specifically, the 6-carbonyl of guanine at positions -9 and -7, the 6-amino group of adenine at position -8, the 5-methyl group of thymine at position -6 and the 2-amino group of guanine at position -5 are identified as primary contacts. The results strongly support the model for duplex recognition in this region of the promoter and suggest that recognition continues along one face of the helix beyond the major groove and into the adjoining minor groove at position -5, where helix melting begins.