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

核肌球蛋白马达的功能：生化和单分子表征。

基本信息

批准号：
MR/M020606/2
负责人：
Christopher Toseland
金额：
$ 29.43万
依托单位：
University of Sheffield
依托单位国家：
英国
项目类别：
Fellowship
财政年份：
2019
资助国家：
英国
起止时间：
2019 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=MR%2FM020606%2F2
关键词：
Function Nuclear Myosin Motors Biochemical

项目摘要

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.

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。这个过程是由基于蛋白质的分子机器协调的，称为RNA聚合酶，它们受到高度调控，以确保正确的表达。RNA聚合酶读取DNA序列以产生信使RNA(MRNA)。信使核糖核酸是一种类似于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，被称为转录因子的额外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。这一规定的崩溃会导致许多并发症，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的发育和干细胞的分化；因此，它是生命从受孕到死亡的各个方面的中心。除了医学影响外，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，为目前对生物技术部门至关重要的生产方法提供低成本、大批量的替代方案。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间。这些调节蛋白本身就是被称为肌球蛋白的分子机器。有趣的是，这些蛋白质通常存在于细胞核外，与肌动蛋白细丝一起运输细胞货物或产生肌肉收缩。虽然肌球蛋白和肌动蛋白都存在于细胞核中，但没有肌动蛋白细丝，这可能表明这两种蛋白可能以完全不同的方式联系在一起。也有证据表明，一些肌球蛋白也可以与DNA结合。因此，有可能在与DNA结合的同时，核肌球蛋白也与RNA聚合酶结合，起到分子钳的作用，将复合体固定在适当的位置。或者，肌球蛋白可能有助于将复合体沿着DNA移动。为了更好地了解这一基本过程，本研究项目将研究核肌球蛋白在转录调控中的作用。分子相互作用的强度将使用允许在毫秒时间尺度上测量微克量蛋白质的技术来确定。使用原子力显微镜和全内反射荧光显微镜等显微技术，也将有可能在单个纳米蛋白质结合DNA并与转录复合体相互作用时可视化它们。最后，将开发一种新的方法来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。这个信号将与RNA聚合酶产生的信使核糖核酸的量相关，从而揭示肌球蛋白马达对这一过程的影响。用这种方法可以确定肌球蛋白是否含有RNA聚合酶、运输聚合酶或组装复合体。这个项目将对这些纳米机器如何调节我们细胞中的基因表达提供最详细的描述。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Autophagy receptor NDP52 alters DNA conformation to modulate RNA polymerase II transcription.

DOI：
10.1038/s41467-023-38572-9
发表时间：
2023-05-18
期刊：
NATURE COMMUNICATIONS
影响因子：
16.6
作者：
dos Santos, Alia;Rollins, Daniel E.;Hari-Gupta, Yukti;McArthur, Hannah;Du, Mingxue;Ru, Sabrina Yong Zi;Pidlisna, Kseniia;Stranger, Ane;Lorgat, Faeeza;Lambert, Danielle;Brown, Ian;Howland, Kevin;Aaron, Jesse;Wang, Lin;Ellis, Peter J. I.;Chew, Teng-Leong;Martin-Fernandez, Marisa;Pyne, Alice L. B.;Toseland, Christopher P.
通讯作者：
Toseland, Christopher P.

Measuring Nuclear Mechanics with Atomic Force Microscopy.

用原子力显微镜测量核力学。