Kinetic modeling and simulation of in vitro transcription by phage T7 RNA polymerase

This study provides a mathematical model of T7 RNA polymerase (T7 RNAP) kin etics under in vitro conditions targeted at application of this model to si mulation of dynamic transcription performance. A functional dependence of t ranscript synthesis rate is derived based on: (a) essential reactant concen trations, including T7 RNAP and its promoter, substrate nucleotides, and th e inhibitory byproduct inorganic pyrophosphate; (b) a distinction among vec tor characteristics such as recognition sequences regulating transcription initiation and termination, respectively; and (c) specific properties of th e nucleotide sequence including both transcript length and nucleotide compo sition. Inactivation kinetics showed a half-life of T7 RNAP activity of 50 min under the conditions applied in vitro using the isolated enzyme. Model parameters and their precision are estimated using dynamic simulation and n onlinear regression analysis. The particular novelty of this model is its c apability to incorporate linear genomic sequence information for simulation of nonlinear in vitro transcription kinetics. (C) 2001 John Wiley & Sons, Inc.