The Kinetics of RNA Polymerase: Visualisation, Simulation, and Inference

Abstract

During transcription, RNA polymerase copies a gene into messenger RNA. This critical biological process is essential for protein production. The main transcription pathway – transcription elongation – involves RNA polymerase translocating the gene from one end to the other, successively catalysing the growth of the RNA chain by incorporating one nucleotide at a time as it translocates. However, this process can go awry due to backtracking or hypertranslocation, where RNA polymerase translocates upstream or downstream from the nascent end of the RNA thus rendering the enzyme inactive. This is associated with transcriptional pausing. Transcription can also go awry from transcriptional slippage, where the gene and the messenger RNA misalign. Slippage can lead to the insertion of uncoded nucleotides into the RNA chain. In this thesis, Bayesian inference is used to explore the kinetics and thermodynamics of transcription elongation and transcriptional pausing. These experiments have provided insights into the parameters governing transcription and its mechanisms. The explanatory powers of various kinetic and quasi-equilibrium models of elongation are compared. A model for the prediction of transcriptional pause sites is presented and its predictive power is evaluated. The role that transcriptional slippage plays in a family of RNA viruses – the Paramyxoviridae – is reviewed and a phylogenetic model detailing the evolutionary history of slippage in this family is presented. Finally, an open-source program – SimPol – is introduced as a means of visualising, simulating, and doing Bayesian inference on RNA polymerases. This is accompanied by Pauser – a program for predicting the locations of pause sites.