Structural and biophysical studies of transcriptional regulatory and anti-termination complexes

The work involves the application of biophysical and structural methods to investigate the mechanism of transcriptional regulation.

Transcriptional anti-termination is a ubiquitous mechanism responsible for regulation and control of gene expression in eubacteria, archaebacteria, eukaryotic viruses and probably also in eukaryotes. The basic tenet of any antitermination mechanism is that the RNA polymerase (RNAP) becomes modified in such a way that it is able to readthrough termination signals allowing it to transcribe into a downstream gene, or further into an operon. The long term goal and objective of this work is to obtain detailed biochemical, biophysical and structural information concerning transcriptional anti-termination complexes in an attempt to provide insight into this important and ubiquitous mechanism of gene regulation. To do this we plan to undertake two main areas of study. The first will concentrate on the identification of components of anti-termination complexes using immunoprecipitation methods combined with mass spectroscopy. The second will focus on the detailed in vitro characterisation of the proteins and complexes involved in antitermination. This will combine structural data obtained from X-ray crystallographic, cryo-electron microscopy and magnetic resonance methods with kinetic and equilibrium binding data obtained from biophysical and biochemical measurements.||Initially, we will focus on the identification and characterisation of the components of antitermination complexes derived from E. coli and M. tuberculosis. However, this will be quickly extended to look at complexes from other organisms such as Mycobacterium leprae and the related but non-pathogenic Mycobacterium phlei and Mycobacterium smegmatis. As rRNA synthesis is intimately linked with bacterial growth rate we will be able to make a valuable comparison between the composition of antitermination complexes from slow growing pathogenic bacteria and the fast growing non-pathogenic varieties. We will also investigate sensor kinase directed antitermination mechanisms, initially concentrating on the proteins from the well-characterised E. coli and B. subtilis Bgl and Sac systems. However, we would also like to look at these systems in slow growing pathogenic mycobacteria, where the control of nutrient uptake is an important issue and sensor kinase directed antitermination mechanisms may well be involved.