Pre-steady-state kinetics of initiation of transcription by T7 RNA polymerase: A new kinetic model

In order to begin to understand the mechanism of the initiation of transcri ption in the model bacteriophage T7 RNA polymerase system, the simplest pos sible reaction, the synthesis of a dinucleotide, has been followed by quenc h-flow kinetics and numerical integration of mechanism specific rate equati ons has been used to test specific kinetic models. Zn order to fit the obse rved time dependence in the pre-steady-state kinetics, a model for dinucleo tide synthesis is proposed in which rebinding of the dinucleotide to the en zyme-DNA complex must be included. Separate reactions using dinucleotide as a substrate confirm this mechanism and the determined rate constants. The dinucleotide rebinding observed as inhibition under these conditions forms a productive intermediate in the synthesis of longer transcripts, and must be included in future kinetic mechanisms. The rate-limiting step leading to product formation shows a substrate dependence consistent with the binding of two substrate GTP molecules, and at saturating levels of GTP, is compar able in magnitude to the product release rate. The rate of product release shows a positive correlation with the concentration of GTP, suggesting that the reaction shows base-specific substrate activation. The binding of anot her substrate molecule, presumably via interaction with the triphosphate bi nding site, likely facilitates displacement of the dinucleotide product fro m the complex. (C) 2001 Academic Press.