Fitting experimental transcription data with a comprehensive template-dependent modular kinetic model.

In the companion article, we developed a modular scheme for representing the kinetics of transcription elongation by RNA polymerase. As an example of how to use these approaches, in this article we use a comprehensive modular model of this sort to fit experimental transcript elongation results obtained on the canonical tR2 template of phage λ by means of complementary bulk gel electrophoresis and surface plasmon resonance assays. The gel electrophoresis results, obtained in experiments quenched at various times after initiation of transcription, provide distributions of RNA lengths as a function of time. The surface plasmon resonance methods were used to monitor increases and decreases in the total mass of transcription elongation complexes in the same experiments. The different measures of transcription dynamics that these methods provide allow us to use them in combination to obtain a set of largely robust and well-defined kinetic parameters. The results show that our modular approach can be used to develop and test predictive kinetic schemes that can be fit to real transcription elongation data. They also suggest that these approaches can be extended to simulate the kinetics of other processes that involve the processive extension or shortening of nucleic acid chains and related systems of sequential branching reaction events.

Monitoring RNA transcription in real time by using surface plasmon resonance.

The decision to elongate or terminate the RNA chain at specific DNA template positions during transcription is kinetically regulated, but the methods used to measure the rates of these processes have not been sufficiently quantitative to permit detailed mechanistic analysis of the steps involved. Here, we use surface plasmon resonance (SPR) technology to monitor RNA transcription by Escherichia coli RNA polymerase (RNAP) in solution and in real time. We show that binding of RNAP to immobilized DNA templates to form active initiation or elongation complexes can be resolved and monitored by this method, and that changes during transcription that involve the gain or loss of bound mass, including the release of the sigma factor during the initiation-elongation transition, the synthesis of the RNA transcript, and the release of core RNAP and nascent RNA at intrinsic terminators, can all be observed. The SPR method also permits the discrimination of released termination products from paused and other intermediate complexes at terminators. We have used this approach to show that the rate constant for transcript release at intrinsic terminators tR2 and tR' is approximately 2-3 s(-1) and that the extent of release at these terminators is consistent with known termination efficiencies. Simulation techniques have been used to fit the measured parameters to a simple kinetic model of transcription and the implications of these results for transcriptional regulation are discussed.