Mechanism of action of small-molecule inhibitors of bacterial gene transcription.

In all living organisms, a multi-part protein, called RNAP, plays a crucial role in an activity of called gene expression. Gene expression is important because it ultimately contributes to the development and survival of the living organism. The RNAP can be regarded as providing a voice to genetic information that, on its own, is silent. Learning how that voice is amplified - and shushed - is a critical stepping stone to many areas of biological and medical research. Thus, the RNAP is a very important protein that directly contributes to the development and survival of all living organisms. To express a gene, the RNAP, must first bind to the DNA-sequence that represents the gene and unwind the paired strands of the DNA-sequence to access the 'template-DNA-strand' to 'read' the information harboured in the gene. Several moving parts of the RNAP, called 'mobile parts' contribute to the DNA binding and DNA unwinding activities of the RNAP, and therefore are often 'control points' for regulating the activity of the RNAP. The activity of the RNAP from bacteria, such as Escherichia coli, is often controlled through the 'mobile parts' of the bacterial RNAP. Intriguingly, viruses which infect and kill bacteria, often do so by making very small proteins, specifically aimed to interfere with the normal functioning of the 'mobile parts' of the bacterial RNAP, which contribute to the DNA binding and DNA unwinding activities. Despite four decades of research on RNAP, very little is known about how the 'mobile parts' of the RNAP contribute to gene expression. The timely work proposed in this application is aimed at studying (i) how two 'mobile parts' of the RNAP contribute to gene expression and (ii) how bacterial virus derived small proteins inhibit the bacterial RNAP. The outcome of the proposed research will have important implications on several aspects of biological and medical research: Understanding how the 'mobile parts' of the bacterial RNAP contribute to gene expression is important to elucidate the process and mechanistic aspects of gene expression in general, since the experimentally easily tractable bacterial RNAP is an excellent model to study gene expression by RNAP in complex organisms, like humans. Understanding how bacterial virus derived small proteins inhibit the bacterial RNAP is significant because they provide a novel point of entry to evaluate targets and generate a knowledge-base for the development of new drugs, which can inhibit the bacterial RNAP and so control the spread of bacterial infectious diseases.

Gene transcription is the most important and first regulatory step in gene expression during developmental and disease states. RNA polymerase (RNAP) is the central enzyme which catalyses transcription and is the target, directly or indirectly, of most regulation of gene expression. RNAPs are multisubunit enzymes and belong to a conserved protein family called 'multisubunit RNAPs'. The bacterial RNAP (bRNAP), particularly the bRNAP from Escherichia coli, is by far the best characterised and the model of choice for detailed mechanistic studies. In the bRNAPs, the two largest subunits, B and B', define the catalytic cleft and contain the catalytic determinants required for transcription. Surrounding the catalytic cleft are structurally flexible and conserved domains of the B and B' subunits, called 'mobile modules', which play important roles during transcription. The precise contribution of these 'mobile modules' to transcription at a genome-wide level has never been investigated. Viruses that infect bacteria (phages) often modulate the bRNAP to serve viral needs by synthesising small molecular weight proteins (called phage inhibitors) that interact and interfere with the normal functioning of the 'mobile modules' of the bRNAP. However, the mechanistic aspects of the interaction between phage inhibitors and the bRNAP have never been systematically explored. Here, the interaction, mode of action and effects of three phage inhibitors on bRNAP from E. coli and the rice pathogen Xanthomonas oryzae will be explored using biochemical and biophysical methods. Microarray technology will be employed to evaluate the (i) contribution of two significant 'mobile modules' of the bRNAP to transcription, and (ii) the effects of phage inhibitors on transcription at a genome-wide level. The outcome of this research will help us understand the mechanistic and regulatory aspects transcription and will benefit those seeking to manage such processes in agriculture, industry and medicine.