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

点击翻译按钮获取中文摘要

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.