Regulation of transcription factor motor activity by autoinhibition and interaction with RNA polymerase

This project concerns the action and control of a molecular motor that is involved in the repair and regulation of genes. Molecular motors break down chemical fuel (typically a molecule called ATP) and the energy that is released is utilised to do some form of physical work. The motor studied in this project is a bacterial protein called Mfd, which uses ATP to power movement along the double stranded DNA molecules that contains the cell's genetic information. This is useful because Mfd can attach itself to another protein complex, called RNA polymerase, and when Mfd moves along the DNA it pushes RNA polymerase forward. RNA polymerase is a key part of the machinery that reads the genetic information held in DNA, and the effect that Mfd has on it depends on the circumstances: sometimes Mfd helps RNA polymerase to start moving along the DNA on its own, and other times Mfd pushes RNA polymerase off the DNA. These interactions can help the cell to control the way in which genes are used and maintained: for example, removal of RNA polymerase from DNA is particularly important if the DNA is damaged because RNA polymerase can prevent the DNA being repaired, which will increase the chance that mutations will arise or the cell will die. Motor proteins must be carefully controlled in order for cells to function correctly . If they are allowed to work in the wrong place or at the wrong time they can interfere with other components of the cell, and if they are allowed to run continuously they will use fuel wastefully, at the expense of other energy-dependent processes. In the case of Mfd, it is only necessary for the motor activity to be turned on when the protein is bound to RNA polymerase. We have discovered that this is ensured by a mechanism that resembles the 'dead mans switch' on an electric lawnmower. Part of the Mfd protein acts as a switch: the motor is turned on when Mfd is held by RNA polymerase, but if RNA polymerase lets go of Mfd the motor turns itself off. The experiments that make up this project will enable us understand how this molecular switch within Mfd works, and how RNA polymerase controls it. The work addresses a fundamental question in molecular biology: there are many similar motors within cells of all kinds, and these often function as part of larger complexes that control their activity by undefined mechanisms. These motors perform different functions from Mfd, but share sufficient common features for lessons learnt from the experimentally tractable Mfd:RNA polymerase system to be useful when trying to understand systems that are more complicated and harder to work with, such as some of the motor-containing complexes that are involved in human health and disease. By gaining a thorough understanding of the ways in which such proteins function we aim to contribute to increased understanding of disease and the design of novel therapeutic strategies.

Motor proteins that couple ATP hydrolysis to movement along nucleic acids play a variety of essential roles in genome function. Often these enzymes act as components of macromolecular complexes, and DNA translocation by the motor protein drives movement of other components of the complex. In order to understand how motor proteins are regulated within multi-protein complexes we are studying a bacterial transcription factor, Mfd, which is a helicase superfamily 2 member that acts upon stalled transcription complexes. This proposal arises out of 3 key observations: the DNA translocation activity of Mfd is inhibited unless the protein is bound to a stalled transcription complex, the inhibition can be alleviated by the deletion of the C-terminal domain of Mfd, and the C-terminal domain interacts in 2-hybrid experiments with the beta flap, a highly conserved flexible region of RNAP that plays a pivotal role in numerous regulatory events during transcription initiation and elongation. These observations indicate that the C-terminal domain of Mfd has an autoinhibitory role, that interaction with a transcription complex alleviates this inhibition, and that the relief of inhibition may be caused by direct binding of the autoinhibitory domain to RNA polymerase. We propose to characterise the mechanism of inhibition and anti-inhibition. We will use directed mutagenesis to introduce substitutions or deletions into regions of Mfd and RNA polymerase that are involved in intra- or inter-molecular interactions. We will then use in vitro assays with purified components to determine the effect of these changes on the DNA translocation activity of Mfd in the presence and absence of transcription complexes. We will also use crosslinking to constrain the autoinhibitory domain in order to determine the degree of flexibility that is required for relief of autoinhibition. Finally, we will characterise the processivity, speed and polarity of DNA translocation by the Mfd protein.