For abstract with figures, click here.
T7 RNA polymerase recognizes a relatively small promoter
extending only seventeen 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) Biochemistry32, 4275-4280; Schick, C. &
Martin, C. T. (1995) Biochemistry34, 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.Return to publication list
