Geometric requirements for gene activation

Controlling access to the information contained in DNA is critical in the development and adaptation of living organisms. The devices that carry out early steps in genetic information flow (gene expression) are small molecular machines often highly regulated by a number of cues. We plan to study one molecular machine at the level of how its use of the energy currency of the cell is converted to a useful output. By analogy, we want to know if a multi-cylinder engine needs to use all its combustion cylinders, can it run on less, and do all connect to the wheels or not. In so doing, we will uncover the design and operating principles of our molecular machine, providing important information about how we might modify and exploit its properties for managing harmful bacteria, creating pathway specific inhibitors and making new molecular devices. The type of device we will study is found in the simplest organisms (bacteria) and amongst the most complex (humans, plants).

Some while ago Bruce Alberts recognised that one major challenge in the life sciences was to achieve an understanding of how the many molecular machines that work to create a living cell would operate. To date we still do not have a comprehensive picture of the varied ways in which the energy currency of the cell is used to power such machines. By asking key questions of one type member bacterial transcription activator whose related proteins are used to control important developmental and adaptive behaviour, we seek to deduce the operating principles of one homo-hexameric ATPase and so gain insight into how the energy coupling reaction between it and the RNA polymerase enzyme is achieved. Our approach is to use protein engineering to organise the hexameric activator protein in ways in which one or more sites for ATPase, regulation of the ATPase, or binding to RNA polymerase is ablated. We will then ask how much and what activities remain, and so deduce the number of sites and their spatial organisation required for full or partial output. Because the activator we study belongs to the largest ATPase super family known, and is present in the three kingdoms of life, results that speak to its operating principles will be of broad significance. In addition, the studies will found advanced biophysical studies using e.g. single molecule FRET, to measure the protein and interaction dynamics at play.The creation of new dimeric forms and hence hexameric forms of the ATPase may also result in new crystal forms being obtained, necessary to advance structural studies of the range of conformations the ATPase can adopt.