Biological functions that depend upon the bridge helix of RNA polymerase

所有生物体中的细胞都通过微小机器的协同作用工作，这些微小机器进行重大转变，以允许生长和适应。这些微小的机器使用生命的积木作为它们的底物，并从中形成有价值的材料，但也会在需要的时候分解这些材料。最终，细胞中的DNA是这些微小机器的蓝图，它将决定机器之间如何相互作用，细胞的组成部分，更重要的是DNA本身。DNA中的遗传信息必须被获取，才能使细胞生长、适应和分化。RNA聚合酶是一种大而复杂的蛋白质，它将DNA复制到RNA模板中，然后从RNA模板中生产蛋白质，从而获得访问权限。在这项建议中，我们试图找出RNA聚合酶的一个保守结构特征如何与RNA聚合酶的其他保守结构特征进行通信，以实现“完整”的功能(就获取DNA中的信息而言)。我们计划研究的耦合相互作用-可以被认为像允许引擎工作和传递力的机械部件的直接耦合-被认为是RNA聚合酶的全部功能所必需的，但到目前为止还没有被证明存在。具体来说，我们将检查(1)所谓的RNA聚合酶切换区和一个称为桥螺旋的长螺旋特征的一端之间的相互作用；(2)确定为什么改变形式的桥螺旋(例如E.ColiF773A突变体)在体外能很好地工作，但不能支持细胞在体内的生长；(3)确定α螺旋桥螺旋中的弯曲点在哪里以及它们对什么是重要的；(4)研究将其导向特定DNA序列的RNA聚合酶相关蛋白质(称为启动子特异性Sigma因子)如何影响桥螺旋的功能。了解细菌RNA聚合酶的工作原理对于了解所有多亚单位RNA聚合酶的功能具有重要意义，也有助于提供知识帮助设计针对转录装置的新的抗微生物药物。我们的最终目标与这一长期目标密切相关，因为我们想要研究的RNA聚合酶的功能似乎非常灵活，可能需要扭结才能发挥作用，这表明它可能会被定制的RNA聚合酶结合化学物质以不利的形态捕获，然后这些化学物质将作为其活性的抑制剂。我们相信，通过了解RNA聚合酶的功能，我们可以为管理感染和疾病的新战略做出贡献，并找到改进生物技术应用的新方法，例如蛋白质和生化产品的生产。

Cells in all living organisms work through the concerted actions of tiny machines that carry out major transformations to allow growth and adaption. These tiny machines using the building blocks of life as their substrates, and form valuable materials from them, but also dismantle such materials when times so require.Ultimately, the DNA in cells is the blue print for these tiny machines, and will set how the machines interact with each other, the cells components, and importantly DNA itself. The genetic information in DNA must be accessed to allow cells to grow, adapt and to differentiate. Access is granted by the regulated activity of the RNA polymerase enzyme, a large and complex protein which copies DNA into an RNA template from which proteins are then produced. In this proposal we seek to work out how one conserved structural feature of RNA polymerase communicates with other conserved structural features of RNA polymerase to achieve 'full' functionality (in terms of accessing the information in DNA).The coupling interactions we plan to study-which can be thought of as being like the direct coupling of mechanical parts that allow engines to work and deliver force- are proposed to be required for the full functionality of RNA polymerase, but to date are not proven to exist. Specifically we will examine (i) interactions between the so called switch regions of RNA polymerase and one end of a long helical feature called the bridge helix; (ii) determine why altered forms of the bridge helix (eg the E. coli F773A mutant) works well in vitro but fails to support in vivo growth of cells;(iii) determine where points of bending in the alpha helical bridge helix are and what they are important for and (iv) look at how an RNA polymerase associated protein that directs it to specific DNA sequences (termed the promoter-specific sigma factor) can impact upon the functioning of the bridge helix. Gaining insights into how bacterial RNA polymerase works has important implications for the understanding of the functioning of all multi-subunit RNA polymerases, as well as in providing knowledge to help in the design of new anti-microbials that target the transcription apparatus.Our final aim is closely related to this longer term goal, since it seems that the feature of the RNA polymerase we wish to study is very flexible and may need to kink to work, indicating it might be captured in an unfavourable configuration by bespoke RNA polymerase-binding chemicals that would then act as inhibitors of its activity. We believe that through knowing how the RNA polymerase enzyme functions we can contribute to new strategies to mange infections and disease, as well as found new ways to improve biotechnological applications, such as protein and bio-chemicals productions.