The RNA polymerase bridge helix and domain communication

Many activities allow cells to grow, remain alive and viable rely on molecules displaying the properties of small machines. The way in which informaton in DNA is accessed relies on one such machine, called RNA polymerase. Although we know the shape and form of RNA polymerase, quite how its parts work together is far less clear. We will be using a program of research to work out how one part-called the Bridge Helix -contributes to RNA polymerase being able to productively interact with DNA. Because RNA polymerases are conserved from man to bacteria, the work is of fundamental importance, and especialy for a knowledge based approach needed for the management of some harmful bacteria.

Understanding the mechanistic basis of gene specific transcription remains a major challenge. Much control of gene expression is exerted at the level of RNA polymerase (RNAP) activity. Yet, the functionality of RNAP is far from understood. Major advances in determining the crystal structures of multisubunit RNAP have been made and RNAP from yeast, archaea and bacteria display a striking structural and sequence conservation. However, the ways in which structurally conserved domains of the RNAP interact for the full function of the RNAP is far from clear, although there is much evidence to suggest the structurally conserved domains are mobile and direct important properties of the enzyme, including promoter recognition, DNA melting and translocation along the DNA. The current proposal focuses on the so called 'bridge helix' of the Escherichia coli RNA polymerase. The functionality of the bridge helix is, so far, only inferred from structural studies and includes interactions with the template DNA, with incoming NTPs and importantly other structurally conserved features of the RNAP. Significantly, the inferred interplay between the bridge helix and the structurally conserved and functionally important domains, known as the 'jaw domain' and the 'trigger loop' remains unproven. Interest in the role of the bridge helix in transcription also arises because its functionality is suggested to be influenced by the binding of at least three different types of antibacterial compounds to the bacterial RNAP. Here, we wish to study the role of the bridge helix by systematic mutagenesis and a thorough characterisation of the mutant RNAPs in a range of assays that report activity and fidelity of the enzyme during transcription initiation, elongation and the influence of the jaw domain on the enzymes functionality. Results will reveal how the bridge helix contributes to RNAP functionality and provide insights into how conserved domains of the RNAP collaborate during transcription.