Misincorporation by wild-type and mutant T7 RNA polymerases: Identification of interactions that reduce misincorporation rates by stabilizing the catalytically incompetent open conformation

We have characterized the misincorporation properties of wild-type (wt) T7 RNAP and of 45 T7RNAP point mutants. The wt enzyme selects strongly against incorporation of an incorrect nucleotide. From the measured rates of misin corporation, an average error frequency of 1 in 2 x 10(4) is estimated. RNA s bearing 3'-mismatches are extended more slowly than correctly paired S'-t ermini, and mismatches one or two bases away from the RNA 3'-end can also s low extension severely even when the 3'-base is correctly paired. Though it has been reported that T7RNAP has a 3' --> 5' nuclease activity, we were u nable to detect ally endogenous T7RNAP RNase activity in elongation complex es. Pyrophosphorolysis was detected but does not appear to contribute to pr oofreading. Therefore, unlike other RNAPs, T7RNAP fidelity appears to depen d entirely on discrimination against incorporation of the incorrect nucleot ide and not on post-misincorporation proofreading. Alanine substitution of the H784 side chain, which interacts with the 3' RNA template base pair, in creases both misincorporation and mismatch extension, while substitutions a t G640, F644, and G645 increase misincorporation, but not mismatch extensio n. The latter three amino acids are in a part of the RNAP which interacts w ith the templating base and with the base immediately 5' to the templating base. Mutation of these amino acids not only increases misincorporation, bu t also eliminates pausing during promoter clearance. The effects of these m utations and the interactions observed in a crystal structure of a transcri bing complex indicate that these mutations disrupt interactions which limit misincorporation rates by stabilizing the catalytically incompetent open c onformation of the RNAP.