Identification of Specific Contacts in T3 RNA Polymerase-Promoter Interactions: Kinetic Analysis using Small Synthetic Promoters
The T7, T3, and SP6 RNA polymerases recognize very similar, yet distinct, promoter sequences. The high homology among the promoter sequences suggests that differential promoter recognition must derive from relatively small changes in the protein. Steady state kinetic analyses of transcription from the T3 consensus promoter and from promoters modified in the region critical to specific recognition reveal details concerning which functional groups contribute to this recognition. Modifications include base pair substitutions, single base substitutions (mismatches), and simple functional group modifications at unique sites in the promoter. The results show that T3 RNA polymerase recognizes the amino group on the nontemplate cytidine in the major groove at position -10, while the identity of the base on the template strand is less critical to binding. In contrast, recognition at position -11 allows a greater range of modifications and seems to have a more complex recognition. The results do not seem to be consistent with a single recognition contact at this position, however, some groups may be ruled out as simple recognition contacts. While major groove modifications weaken binding at positions -10 and -11, the removal of an exocyclic amino group from the minor groove at either position does not disrupt binding, further supporting a model for promoter recognition in which the enzyme binds to one face of closed duplex DNA in this region. The effects of these changes in the DNA structure on the kinetics of initiation are compared to complementary results from the T7 system.