The overall objective of this research is to elucidate the catalytic mechanism of DNA-dependent RNA polymerase. In order to obtain such information, we propose to continue and extend our studies of T7 RNA polymerase in which functionally significant residues have been altered. By site-directed mutagenesis, we have mutated five sites which are strongly conserved among RNA and DNA polymerases and shown that D537 and D812 are essential for activity, and K631, Y639 and H811 are functionally significant. The role in the polymerase mechanism of each of these functional groups, and others to be identified by further mutagenesis, will be determined. Each step in the addition of single nucleotides to initiation and elongation complexes will be analyzed by pre-steady-state experiments utilizing abortive initiation, quench-flow, pyrophosphorolysis and pyrophosphate exchange, and fluorescence-detected stopped flow. Thio analogs of the nucleotides will be used to help distinguish between chemical and non-chemical (conformational changes and product dissociation) steps. A detailed comparison between the kinetic and equilibrium parameters for the wild-type and mutant enzymes will permit assignment of specific roles to each mutated side-chain. Specific experiments will test mechanistic hypotheses, e.g. the possibility that the two critical Asp residues bind divalent cations in a two-metal-ion mechanism as in DNA polymerase. Binding of Mn2+ to wild-type and mutants with replacements at D537 and D812 will be measured by EPR and flow dialysis. Mn2+in the presence of HCO3 will be used to catalyze the H202 oxidation of side chains near the metal binding site. These residues can be labeled by reduction with NaB3H4 and identified after digestion and peptide separation. Co2+ nucleotides will be crosslinked to wild-type and mutants by H202 oxidation. Further sites critical for RNA polymerase activity will be identified by cassette mutagenesis. It is these presently unidentified sites which are most likely to be unique to RNA polymerase as opposed to DNA polymerase. CD and fluorescence will be used to compare the conformation and stability of the mutants with the wild-type enzyme. Photoaffinity labeling with nascent RNAs in which 8-N3ATP has been incorporated will be used to identify regions of the enzyme with which the product RNA interacts.
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