Pre-steady-state and steady-state kinetic studies on transcription initiation catalyzed by T7 RNA polymerase and its active-site mutants K631R and Y639F.

The kinetic mechanism of transcription initiation was studied under conditions that allow a single nucleotide addition to an initiating dinucleotide without interference of enzyme-DNA dissociation or protein recycling. Pre-steady-state kinetic studies have provided polymerization rate constants of 3.9, 5.9, and 3.9 s-1, reverse polymerization rate constants of 3.2, 2.1, and 2.8 s-1, and dissociation constants for the incoming nucleotide of 26, 49, and 24 microM at 21 degreesC, respectively, for the wild type and its active-site mutants K631R and Y639F. The results suggest a model in which K631 interacts with the phosphate group(s) of the incoming substrate. The internal equilibrium constants for the bound species are close to unity, consistent with the values for other phosphoryl transfer enzymes. The rate constants for chemical bond formation are at least 50 times higher than the rate constants for product dissociation. The product release rate constants, k3, are comparable to the steady-state rates, suggesting that the rate-determining step for all three enzymes may be a product dissociation step. The existence of two possible conformers E and E' that are in rapid equilibrium is postulated, to reconcile reduced burst sizes with full activity of the mutant enzymes. Both forms can form the quaternary complex, but only the E form is capable of catalyzing phosphodiester bond formation. The fraction of the catalytically active E form varies from essentially 100% for the wild type to 38 and 32% for the mutants K631R and Y639F, respectively. Upon entering the elongation phase, the E form becomes the dominant form in all three enzymes, leading to comparable rates of elongation for the wild type and Y639F mutant. The rate of synthesis of long transcripts is markedly diminished for the K631R mutant due to decreased processivity.