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

基因表达是指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。这一过程是由基于蛋白质的分子机器精心安排的，称为RNA聚合酶，它受到高度调节以确保正确的表达。RNA聚合酶读取DNA序列生成信使RNA （mRNA）。mRNA是一种类似于DNA的分子，被细胞机器读取，将序列翻译成蛋白质。当需要表达时，另一种叫做转录因子的蛋白质会激活这些机器。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。这种调节的破坏会导致许多并发症，包括发育障碍，最明显的是癌症的形成。此外，表达的变化控制着胚胎发育和干细胞分化；因此，从受孕到死亡，它对生命的各个方面都至关重要。除了医学意义之外，了解这一重要过程可以增强无细胞蛋白质生产系统，为当前的生产方法提供低成本、大批量的替代方案，这对生物技术部门非常重要。最近，在细胞核中发现了新的调控蛋白，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凝蛋白的分子机器。有趣的是，这些蛋白通常在细胞核外发现，与肌动蛋白丝一起运输细胞货物或产生肌肉收缩。虽然肌凝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可能以完全不同的方式结合。也有证据表明，一些肌凝蛋白也可以与DNA结合。因此，核肌凝蛋白在与DNA结合的同时，也有可能与RNA聚合酶结合，起到分子夹子的作用，将复合物固定在适当的位置。或者，肌凝蛋白可以帮助复合物沿着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凝蛋白在调节转录中的作用。分子相互作用的强度将使用技术来确定，这种技术可以在毫秒时间尺度上以微克量的蛋白质进行测量。使用显微镜技术，如原子力显微镜和全内反射荧光显微镜，也将有可能看到单个纳米级蛋白质结合DNA并与转录复合物相互作用的过程。最后，将开发一种新的分析方法，以便直接实时测量转录过程。这需要开发一种生物传感器，一种与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数量相关，从而揭示肌凝蛋白马达对该过程的影响。用这种方法可以确定肌凝蛋白是否持有RNA聚合酶，运输聚合酶或组装复合物。这个项目将提供这些纳米机器如何调节我们细胞中的基因表达的最详细的描述。

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.