Function of Nuclear Myosin Motors: A Biochemical and Single Molecule Characterization.

Gene expression, the transfer of the genetic code into cellular proteins is one of the most fundamental processes in living cells. This process is orchestrated by protein-based molecular machines, called RNA polymerases, which are highly regulated to ensure correct expression. RNA polymerases read the DNA sequence to generate messenger RNA (mRNA). mRNA, a molecule similar to DNA, is read by the cellular machinery to translate the sequence into a protein. Additional proteins called transcription factors activate these machines when expression is required. Our cells have evolved elaborate regulation mechanisms to control these molecular machines. A breakdown in this regulation leads to numerous complications including development disabilities and most notably cancer formation. Furthermore, changes in expression control embryonic development and stem cell differentiation; thus it is central to all aspects of life from conception to death. Aside from the medical implications, understanding this vital process could lead into enhancements cell-free protein production systems providing low cost, high volume alternatives to current methods of production which are important for biotechnological sectors.
Recently, new regulatory proteins have been discovered in the nucleus, the compartment in the cell which stores genetic material. These regulatory proteins are themselves molecular machines called myosins. Interestingly, these proteins are usually found outside the nucleus transporting cellular cargo or generating muscle contraction in association with actin filaments. While myosin and actin are both present in the nucleus, there are no actin filaments, which could indicate that the two proteins may associate in a completely different manner. There is also evidence that some myosins can also bind to DNA. Therefore, it could be possible that, while bound to DNA, nuclear myosins also bind to the RNA polymerase, acting as molecular clamps and holding the complex in place. Alternatively, myosin may help to move the complex along DNA.
With the aim of gaining a better understanding of this fundamental process, this research project will investigate the role of nuclear myosins in regulating transcription. The strength of molecular interactions will be determined using techniques which allow measurements on millisecond time-scales with micro-gram quantities of protein. Using microscopy techniques such as atomic force microscopy and total internal reflection fluorescence microscopy, it will be also possible to visualise the individual nanoscopic proteins as they bind DNA and interact with the transcription complex. Finally, a novel assay will be developed in order to directly measure the process of transcription in real-time. This requires the development of a biosensor, a protein which will generate a fluorescent signal when binding to mRNA. This signal will correlate with the amount of mRNA produced by the RNA polymerase and therefore reveal the effect of the myosin motors on the process. With this method it can be determined whether the myosin holds the RNA polymerase, transports the polymerase or assembles the complex. This project will provide the most detailed description of how these nanoscopic machines regulate gene expression in our cells.

A combination of single molecule (Total internal reflection fluorescence and atomic force microscopy) and transient kinetics will be used to investigate the function of nuclear myosin I and VI, and their roles in transcription. This will provide a detailed insight into the regulation and mechanism of gene expression.
An overriding hypothesis is that myosins are anchored to DNA and aid RNA polymerase (RNAP) activity through interactions with monomeric actin (short polymeric) on the RNAP complex. NMI will be characterised to determine its motor properties in relation to its cytoplasmic isoform (Myosin Ic). NMI and Myosin VI interactions with DNA and monomeric actin will then be explored. Transient kinetics will be able to determine the binding conditions on millisecond time scales with microgram quantities of protein. And single molecule imaging will allow visualisation of how the motors bind and potentially move DNA. This combined approach will determine the strength of the interactions, identify binding sites, how binding occurs and effects upon the motor proteins and reveal their mechanism of action. Also, through single molecule tracking of fluorescently labelled myosin and DNA, it will then be possible to determine if the motors can generate motility when bound to DNA.
Investigations will then focus on the transcription complex. Kinetic and hydrodynamic approaches will be used to measure interactions between myosin and RNAP. While single molecule methods will localise myosin on RNAP. Finally, a novel assay will be developed to measure RNAP activity. The assay will require the design of a fluorescent mRNA biosensor, which will bind rapidly and specifically to mRNA allowing the activity to be tracked in real-time. This sensitive approach will allow the effect of myosin motors to be explored in a highly controlled environment. This effect can then be related to the characterisation measurements to understand how the myosin triggers responses during transcription.

The main objectives are to characterize the activity of nuclear myosin motors and understand their role in RNA transcription. This work is particularly relevant to scientists working in the fields of myosin motors, RNA transcription, nuclear organisation, DNA-protein interactions, molecular machines, molecular motor proteins, bio-nanotechnology and single-molecule biophysics. This impact will be immediate: i.e. within the lifetime of the grant and beyond. The research will provide a foundation governing the activity of these molecular motors and their roles in gene expression.
On a longer timescale, secondary research which would be supported by this work will be directly translatable in the fields of control and regulation of gene expression. This has direct implications for cancer, aging and stem cell research. Overall, through fundamental research this will improve the health and wellbeing of society. Biotechnological applications may also arise for the enhancement of mammalian protein expression systems or even cell-free expression systems based upon a greater understanding of the mechanism driving these processes. Furthermore, through the investigation and understanding of these cellular machines, the construction of novel nano-machines becomes possible. Finally, the mRNA biosensor would be an important tool which could be optimise for pharmaceutical and medical testing. Overall, this would be beneficial for the public health, pharmaceutical and biotechnology, animal and plant health sectors and consequently to the economic competitiveness of the UK. Biosensors for medical testing/screening purposes, especially those engineered for RNA can provide methods for simple/quick RNA virus (SARS, Influenza, Hep C and retroviruses such as HIV) detection which can benefit global health.
High quality training in various disciplines (as outlined in the Case for Support) will produce highly skilled researchers. This is beneficial to UK science, health and pharmaceutical sectors, which aids the economic output of the UK. This will help to maintained the UK's world leading medical research sector.
Through a program of engagement with the public, this work will also benefit the wider UK community by communicating the excitement of modern interdisciplinary biology, including the state-of-the-art single molecule techniques. This is particularly important for younger people who may be considering pursuing a career in science. This will be mostly through School Days and the Nuffield placement scheme. The University of Kent has an extensive portfolio of mechanisms for public engagement, as well as organising public engagement activities.