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

Lead Research Organisation: University of Bristol
Department Name: Biochemistry


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.

Technical Summary

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.


10 25 50
Description From original final report form (2011):

The Mfd protein displaces stalled transcription complexes using an ATP-dependent DNA translocation activity. This activity is inhibited in the isolated protein, but activated when Mfd binds to RNA polymerase (RNAP). Such context-specific control is likely to be widespread amongst the many motor proteins that act as components of complexes involved in genome function, and our aim was to understand the molecular details of the process.
We used mutagenesis to examine how domain 7 (D7) of Mfd causes autoinhibition of the protein. We found that a key factor in the process is the intramolecular interaction of D7 with domain 2 (D2: located in the N-terminal region of the protein). We have shown that Mfd that lacks the N-terminal region behaves very similarly to the previously-characterised Mfd mutant that lacks D7 e.g. it shows elevated motor activity in the absence of RNAP. Furthermore, we have identified amino acid substitutions within the D2:D7 interface that relieve the autoinhibition of the protein and cause changes in the susceptibility of the amino acid linker that joins the N- and C-terminal regions of Mfd to proteolysis. The model that has emerged from this work is that (i) interaction between the regions of Mfd that flank the motor domains forms a rigid clamp that constrains the movement of the motor domains and so inhibits their activity, and (ii) interaction with RNAP breaks the interdomain interaction and allows Mfd to adopt a more open and flexible conformation. However, our work also suggests that the clamp formation is not the only mechanism by which D7 inhibits Mfd. We have isolated a mutant Mfd protein that is mutated in the region that links D7 directly to the C-terminal motor domain. The protease-cleavage pattern of this mutant differs from those described above, and it has an increased affinity for DNA. Full-length Mfd carrying this substitution has the unusual properties of being able to destabilise transcription initiation complexes (which are not substrates for wild-type Mfd) and to displace stalled transcription elongation complexes in the absence of the known Mfd:RNAP contacts.
At the beginning of this work we had identified an interaction between D7 of Mfd and a region of RNAP called the beta flap. As the beta flap plays regulatory roles in transcription elongation and termination it was important to determine the functional consequences of this interaction. We made RNAPs that lacked all or part of the beta flap, and investigated the effect of Mfd using templates built to function with truncated RNAP. We found that deletion of the beta flap does not prevent wild-type Mfd from displacing RNAP, and does not prevent activation of the DNA translocation activity. We conclude that the interaction between domain 7 of Mfd and the beta flap of RNAP is not an essential component of the mechanism by which RNAP relieves the autoinhibition of Mfd. We also showed that this interaction is not responsible for the ability of certain paused transcription complexes to resist the action of Mfd.
We examined how far and how fast Mfd can move on DNA once it has been activated by RNAP by analysing the movement of Mfd between two points on substrates of differing length. Attempts to measure the speed of translocation were inconclusive (the question is now being addressed via a collaboration with a group conducting single molecule experiments), but surprisingly we found that Mfd can travel at least 500 bp from the point at which it is activated. This is considerably further than would be necessary to push RNA polymerase off the DNA, and led us to reconsider how DNA damage might be located by the repair machinery during transcription-coupled repair. Experiments by other BBSRC-funded workers in the lab suggest that this extended translocation can lead to rapid repair of DNA lesions located well downstream of a stalled RNAP.

Key findings:
We identified the intramolecular interactions that are responsible for the autoinhibition of Mfd motor activity in the absence of RNA polymerase (RNAP). Our results support a model in which the N- and C-terminal regions of the protein form a rigid "clamp" around the translocase domains in the isolated protein, and a substantial conformational change occurs upon activation (Murphy 2009, Manelyte 2010).
We showed that Mfd can translocate for at least 500 bp after interacting with RNAP. This processivity is far greater than anticipated, and suggests that DNA translocation by Mfd is likely to be important for the location of DNA damage after RNAP has been displaced from DNA (manuscript in preparation).
We showed that the beta flap of RNAP (a critical domain for many transcriptional processes) is dispensable both for the displacement of RNAP by Mfd and for the resistance of some stalled transcription complexes to Mfd (manuscript in preparation).
Exploitation Route The research project concerned a mechanism for the maintenance of genome stability in bacteria. Consequently, our results are of use to researchers studying processes arising from genome instability (such as mutation giving rise to antibiotic resistance, or altered properties of pathogenic or commercially important bacteria), and to researchers and industrial labs aiming to engineer, or design from scratch, the genomes of cells to reliably perform specific functions (such as the production of high value chemicals). Since the research project was completed it became apparent that the protein at the centre of our research was, counter-intuitively, involved in promoting mutation to antibiotic resistance in the important food-borne pathogen Campylobacter jejunii. Our discoveries about how the protein works are helpful in thinking about how to reduce the rise of antibiotic resistance in this organism.
Sectors Agriculture, Food and Drink,Education,Manufacturing, including Industrial Biotechology

Description This research project related to the understanding of a fundamental process in biochemistry. It is expected that most short term impact of this research will relate to its influence on the work of other academic laboratories, which are not considered in this section. In the long term this research is anticipated to have direct or indirect impact on a number of fields outside academia (see RCUK Key Findings), but the measurable societal impact at present has come through public engagement activities (Open days, Science cafe, school visits, press release).
First Year Of Impact 2008
Sector Education
Impact Types Cultural,Societal

Description Press release (Manelyte paper) 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Not monitored.
Year(s) Of Engagement Activity 2010
Description University of Bristol Open Days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact One-to-one discussions with members of the public about our research, biochemical sciences generally, and other aspects of University study.

It is difficult to report specific impacts. I have no doubt that the discussions influenced the life choices of many of the young people that I spoke with.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011,2012,2013,2014,2015