Road Rules for Traffic on DNA: Systematic Analysis of Gene Regulation By Encounters

Textbook depictions of the transcription of a gene show RNA polymerase binding to the DNA at the promoter, initiating transcription and then transcribing uneventfully across the gene until reaching a terminator sequence and falling off the DNA. However, this picture of the DNA as a freeway for RNA polymerase does not apply within a cell, where the DNA is more like a crowded one-lane two-way street. As an RNA polymerase makes its way along the DNA, its progress can be affected by a myriad of proteins bound to the DNA, by difficult DNA sequences that make it pause, and also by other traffic - RNA polymerases moving either in the opposite direction or more slowly in the same direction. The outcomes of these collisions are crucial in gene regulation, yet the factors that determine the ‘winner’ – whether the DNA-bound protein blocks transcription by RNA polymerase or whether the polymerase dislodges the protein and interferes with its function – are unknown.

Here we integrate in vivo experiments in E. coli, using well-defined genetic components, with mathematical modelling to study the factors which influence the outcome of these interactions. Construction of a modular, chromosomally integrated single-copy chassis allows us to systematically vary parameters such as RNA polymerase density, promoter-roadblock spacing, and roadblock binding affinity. This combined approach allows us to evaluate the significance of RNA polymerase - roadblock encounters, the factors that affect them and may provide new tools for the manipulation of gene expression.