Tests of a model for promoter recognition by T7 RNA polymerase: thymine methyl group contacts.

Abstract

The DNA-dependent RNA polymerase from bacteriophage T7 is highly specific for a 17 base promoter sequence. Interactions between T7 RNA polymerase and its promoter DNA have been probed using modified oligonucleotides and a steady-state kinetic assay. The incorporation of deoxyuridine in place of thymidine at individual sites in the promoter sequence results in the replacement of an exocyclic methyl group by hydrogen (effectively removing the thymine methyl). This substitution has been placed individually at each of the thymines in the T7 consensus promoter. Many of these substitutions do not affect binding or catalysis; however, the thymine methyl group at position-6 is critical to recognition. The kinetic parameter Km increases approximately 10-fold while kcat is only slightly affected, suggesting that this thymine methyl is critical to binding specificity, but not to the kinetics of initiation. Two methyl groups near the start site on the template strand (at positions -1 and -3) also contribute to promoter specificity, while nearby methyl groups on the nontemplate strand do not. The implications of these results are discussed with respect to recent models for promoter binding.