Bacteriophage T7 RNA polymerase and its active-site mutants. Kinetic, spectroscopic and calorimetric characterization.

Abstract It has been demonstrated that the amino acids Asp537, Asp812, Lys631, His811 and Tyr639 are involved in bacteriophage T7 RNA polymerase catalysis. In the present paper, we report kinetic, spectroscopic and calorimetric characterization of the wild-type and mutant T7 RNA polymerases generated at these five loci (D537N, E; K631M, R; Y639F, S, A, W; H811Q, A; D812N, E). The wild-type enzyme has a substantial amount of secondary structure as determined by CD analysis (α-helix, 43%; β-sheet, 14%; β-turn, 25%; unordered, 18%). The CD spectra of 12 mutants at five loci are very similar to that of the wild-type, except for the mutant Y639W. Within experimental error, the thermal transition temperatures measured by CD and DSC as well as the λ max values of the fluorescence spectra were the same for the wild-type and all of the mutants. Therefore, the overall folding and stability of the mutant enzymes are very similar to those of the wild-type enzyme, although small local conformational changes cannot be excluded. For the synthesis of the pentamer pppGGACU, the mutants D537E and D812E showed an approximately two-to threefold decrease in ( k cat ) app and an approximately two- to threefold increase in ( K m ) app , relative to the wild-type, in contrast to the mutants D537N and D812N which exhibited no detectable activity. The mutant K631R showed a sevenfold reduction in ( k cat ) app and a two- to threefold increase in ( K m ) app supporting our earlier observation with the mutant K631M that Lys631 may be involved in phosphodiester bond formation. The mutant Y639S can systhesize the trimer GGA with an approximately 50-fold decrease in ( k cat ) app and a tenfold increase in ( K m ) app relative to the wild-type, underlining the importance of the phenyl ring of Tyr639. The mutant H811A, in which the side-chain at position 811 is incapable of forming a hydrogen bond, can synthesize the trimer GGA with an approximately tenfold decrease in ( k cat ) app and an approximately 35-fold increase in ( K m ) app . Thus, either the hydrogen-bonding capacity of this residue is non-essential or some other group can functionally substitute for the His811 side-chain. The wild-type enzyme showed significant effects of the base position in the sequence on the apparent binding constants for the NTPs. The kinetics of GpG-primed trimer, tetramer and pentamer synthesis on three 22 bp templates were investigated for the wild-type and mutant enzymes with measurable activity. The rate of oligonucleotide synthesis depends strongly on the message sequence, and abortive termination occurs more frequently following UMP incorporation. For a given template, the rate of synthesis by the wild-type and all the mutants is trimer>pentamer (run-off transcript)>tetramer. The effect of the 5′-triphosphate in the initiating nucleotide is minimal for the wild-type and mutant enzymes. The apparent p K values obtained from the high-pH side of the pH activity profiles of the wild type and the mutants K631R and Y639F are the same within experimental error, implying that an amino acid(s) other than Lys631 or Tyr639 is responsible for the high-pH side of the pH-activity profile.