Promoter specificity determinants of T7 RNA polymerase

The high specificity of T7 RNA polymerase (RNAP) for its promoter sequence is mediated, in part, by a specificity loop (residues 742–773) that projects into the DNA binding cleft (1). Previous work demonstrated a role for the amino acid residue at position 748 (N748) in this loop in discrimination of the base pairs (bp) at positions −10 and −11 (2). A comparison of the sequences of other phage RNAPs and their promoters suggested additional contacts that might be important in promoter recognition. We have found that changing the amino acid residue at position 758 in T7 RNAP results in an enzyme with altered specificity for the bp at position −8. The identification of two amino acid:base pair contacts (i.e., N748 with the bp at −10 and −11, and Q758 with the bp at −8) provides information concerning the disposition of the specificity loop relative to the upstream region of the promoter. The results suggest that substantial rearrangements of the loop (and/or the DNA) are likely to be required to allow these amino acids to interact with their cognate base pairs during promoter recognition.