Biological functions that depend upon the bridge helix of RNA polymerase

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Cells in all living organisms work through the concerted actions of tiny machines that carry out major transformations to allow growth and adaption. These tiny machines using the building blocks of life as their substrates, and form valuable materials from them, but also dismantle such materials when times so require.Ultimately, the DNA in cells is the blue print for these tiny machines, and will set how the machines interact with each other, the cells components, and importantly DNA itself. The genetic information in DNA must be accessed to allow cells to grow, adapt and to differentiate. Access is granted by the regulated activity of the RNA polymerase enzyme, a large and complex protein which copies DNA into an RNA template from which proteins are then produced. In this proposal we seek to work out how one conserved structural feature of RNA polymerase communicates with other conserved structural features of RNA polymerase to achieve 'full' functionality (in terms of accessing the information in DNA).The coupling interactions we plan to study-which can be thought of as being like the direct coupling of mechanical parts that allow engines to work and deliver force- are proposed to be required for the full functionality of RNA polymerase, but to date are not proven to exist. Specifically we will examine (i) interactions between the so called switch regions of RNA polymerase and one end of a long helical feature called the bridge helix; (ii) determine why altered forms of the bridge helix (eg the E. coli F773A mutant) works well in vitro but fails to support in vivo growth of cells;(iii) determine where points of bending in the alpha helical bridge helix are and what they are important for and (iv) look at how an RNA polymerase associated protein that directs it to specific DNA sequences (termed the promoter-specific sigma factor) can impact upon the functioning of the bridge helix. Gaining insights into how bacterial RNA polymerase works has important implications for the understanding of the functioning of all multi-subunit RNA polymerases, as well as in providing knowledge to help in the design of new anti-microbials that target the transcription apparatus.Our final aim is closely related to this longer term goal, since it seems that the feature of the RNA polymerase we wish to study is very flexible and may need to kink to work, indicating it might be captured in an unfavourable configuration by bespoke RNA polymerase-binding chemicals that would then act as inhibitors of its activity. We believe that through knowing how the RNA polymerase enzyme functions we can contribute to new strategies to mange infections and disease, as well as found new ways to improve biotechnological applications, such as protein and bio-chemicals productions.