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

基因表达，将遗传密码转移到细胞蛋白质中是活细胞中最基本的过程之一。这个过程是由蛋白质为基础的分子机器，称为RNA聚合酶，高度调控，以确保正确的表达。RNA聚合酶读取DNA序列以产生信使RNA（mRNA）。mRNA是一种类似于DNA的分子，由细胞机器读取，将序列翻译成蛋白质。当需要表达时，称为转录因子的其他蛋白质激活这些机器。我们的细胞已经进化出复杂的调节机制来控制这些分子机器。这一规则的崩溃导致许多并发症，包括发育障碍，最明显的是癌症的形成。此外，表达的变化控制胚胎发育和干细胞分化;因此，它是从受孕到死亡的生命各个方面的核心。除了医学意义外，了解这一重要过程可能会导致增强无细胞蛋白质生产系统，为目前的生产方法提供低成本，高产量的替代品，这对生物技术部门很重要。最近，在细胞核中发现了新的调节蛋白，细胞核是细胞中储存遗传物质的隔室。这些调节蛋白本身就是称为肌球蛋白的分子机器。有趣的是，这些蛋白质通常在细胞核外发现，运输细胞货物或产生与肌动蛋白丝相关的肌肉收缩。虽然肌球蛋白和肌动蛋白都存在于细胞核中，但没有肌动蛋白丝，这可能表明这两种蛋白质可能以完全不同的方式结合。还有证据表明，一些肌球蛋白也可以与DNA结合。因此，核肌球蛋白在与DNA结合的同时，也可能与RNA聚合酶结合，充当分子钳并将复合物固定在适当的位置。或者，肌球蛋白可能有助于使复合物沿着DNA移动。为了更好地理解这一基本过程，本研究项目将研究核肌球蛋白在调节转录中的作用。分子相互作用的强度将使用允许用微克量的蛋白质在毫秒时间尺度上测量的技术来确定。使用显微镜技术，如原子力显微镜和全内反射荧光显微镜，也将有可能可视化单个纳米级蛋白质，因为它们结合DNA并与转录复合物相互作用。最后，将开发一种新的检测方法，以直接实时测量转录过程。这需要开发一种生物传感器，一种当与mRNA结合时会产生荧光信号的蛋白质。该信号将与RNA聚合酶产生的mRNA的量相关，因此揭示了肌球蛋白马达对该过程的影响。用这种方法可以确定肌球蛋白是否携带RNA聚合酶，运输聚合酶或组装复合物。该项目将提供这些纳米机器如何调节我们细胞中基因表达的最详细描述。

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.