PROMOTER SPECIFICITY DETERMINANTS OF T7 RNA-POLYMERASE

The high specificity of T7 RNA polymerase (RNAP) for its promoter sequ ence is mediated, in part, by a specificity loop (residues 742-773) th at 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 promote rs suggested additional contacts that might be important in promoter r ecognition. We have found that changing the amino acid residue at posi tion 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 specific ity loop relative to the upstream region of the promoter. The results suggest that substantial rearrangements of the loop (and/or the DNA) a re likely to be required to allow these amino acids to interact with t heir cognate base pairs during promoter recognition.