Kinetic mechanism of transcription initiation by bacteriophage T7 RNA polymerase.

Abstract

The kinetic mechanism of transcription initiation by bacteriophage T7 RNA polymerase was investigated using transient state kinetic methods. Transcription by bacteriophage T7 RNA polymerase occurs in three stages consisting of initiation, promoter clearance, and elongation. Abortive products, up to 6-8-mer, were synthesized during the initiation phase; the transition from initiation to elongation occurred between the synthesis of 6-8-mer and 11-12-mer, and the processive elongation phase began after the synthesis of 12-mer RNA. Our results show that the synthesis of elongation product from the phi 10 promoter is limited both by the efficiency of initiation and by the frequency at which the polymerase escapes the promoter. Studies with heparin trap suggest that the polymerase maintains contact with the promoter region during multiple turnovers of abortive RNA synthesis; thus, the polymerase does not completely dissociate from the promoter after each event of abortive RNA synthesis. The pre-steady-state kinetics of RNA synthesis indicate that initiation occurs at a rate constant (3.5 s(-1)) that is about 30 times faster than the steady-state rate constant of RNA synthesis (0.1 s(-1)). The steady-state rate constant of RNA synthesis is limited largely by the cycling of the RNA polymerase, whereas initiation is limited by the formation of pppGpG, the first RNA product. We show that the synthesis of pppGpG is not limited by steps associated with GTP binding, DNA binding, or the melting of the promoter DNA. Instead, the kinetic results indicate that initiation at the phi10 promoter is limited either by the first phosphodiester bond formation step or more likely by a conformational change prior to pppGpG formation. Such a conformational change could play a role in proper alignment of the initiating and elongating NTPs for efficient phosphodiester bond formation and in maintaining the fidelity of RNA synthesis.