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

Abstract

This study provides a mathematical model of T7 RNA polymerase (T7 RNAP) kinetics under in vitro conditions targeted at application of this model to simulation of dynamic transcription performance. A functional dependence of transcript synthesis rate is derived based on: (a) essential reactant concentrations, including T7 RNAP and its promoter, substrate nucleotides, and the inhibitory byproduct inorganic pyrophosphate; (b) a distinction among vector characteristics such as recognition sequences regulating transcription initiation and termination, respectively; and (c) specific properties of the nucleotide sequence including both transcript length and nucleotide composition. 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 nonlinear regression analysis. The particular novelty of this model is its capability to incorporate linear genomic sequence information for simulation of nonlinear in vitro transcription kinetics. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 72: 548–561, 2001.

The Presence of an RNA:DNA Hybrid That Is Prone to Slippage Promotes Termination by T7 RNA Polymerase

“…As the concentration of CTP is decreased the abundance of slipped products increases dramatically. At concentrations of CTP that correspond to reported values of K M app for CTP (∼30 µM 22 ) most of the transcripts from the U 8 template are larger than expected (Fig. 2c).…”

“…Transcript slippage vs the concentration of CTP. Templates having the variable regions indicated were transcribed in the presence of serial two-fold dilutions of CTP (for each template, left to right: 800, 400, 200, 100, 50, 25, 12.5µM); the range of previously reported values for K M app [CTP] 22 are indicated by the bar below the image.…”

Synthetic in vitro transcriptional oscillators

“…Despite the introduction of short degradation products, the extended model fits could not capture quantitative aspects for many reaction conditions. Poor fits can be expected for the initial part of fluorescence measurement because of a 'burst phase' in enzyme kinetics (Jia and Patel, 1997) and for the final part of fluorescence measurement because of buffer exhaustion, NTP depletion, build-up of waste products, product inhibition (Arnold et al, 2001), and degradation of enzyme functionality-none of which were modeled. Rigorous mechanistic modeling that explicitly considers these factors may improve quantitative accuracy for our in vitro transcription systems.…”

Analysis-based parameter estimation of an in vitro transcription-translation system

“…Several examples for modeling of the transcription and translation processes can be found in the literature, ranging from coarse grain [11], [21] to very detailed [9], [1] focusing on different aspects of gene expression. Our aim here is to analyze and improve a previously proposed ODE-based model describing transcription and translation in a cell-free experimental environment using real measurement data.…”