Structures and Mechanisms of RNA polymerase inhibition and activation

RNA聚合酶是一种基本的细胞机制，负责将储存在DNA中的遗传信息转化为另一种称为RNA的遗传分子，然后可以转化为蛋白质或以另一种调节能力发挥作用。DNA中的核糖核酸信息发生在一个复杂的、高度受控的过程中，称为基因转录，核心分子机制RNAP酶从细菌到人类都是保守的。基因转录是发育过程中高度调控的事件，是所有已知生命系统对生长和环境刺激的主要反应。尽管在理解RNAP作为酶的功能方面取得了重大进展，包括2006年诺贝尔化学奖认可的工作，但RNAP本身如何受到信号特殊细胞状态和事件的因素的控制，仍然知之甚少。我们使用细菌RNAP及其主要变体sigma因子sigma 54作为简化的模型系统来研究RNAP如何保持在抑制状态，以及激活蛋白如何从转录开始的地方远程作用，以称为ATP的高能分子的形式利用细胞能量，将RNAP从无活性酶转化为转录活性酶。我们刚刚确定了RNAP-sigma 54的晶体结构，因此我们现在可以详细了解RNAP-sigma 54的外观。我们的结构解释了sigma 54如何维持RNAP处于抑制状态。此外，我们发现我们看到的许多抑制策略在某种程度上被其他细菌和真核生物因子共享，并揭示RNAP中存在保守的热点，这些热点在不同程度上被不同的元件和转录因子靶向以微调转录抑制。在目前提出的这项研究中，我们计划利用我们新获得的知识，经验和已经产生的试剂来解决有关激活剂如何缓解这种抑制状态的基本问题。这很可能揭示人类，植物和动物中的RNAP是如何被激活的。此外，我们希望利用抑制状态的结构特征来设计新的抗生素，通过攻击细菌RNAP酶上的重要位点和表面来抑制基因转录，这些位点和表面以前没有被靶向用于药物治疗。抑制细菌RNAP，从而抑制基因转录，是一种有效的抗生素策略，例如在控制TB感染中，因此我们的工作应该为有效的抗生素开发提供新的途径，因为在这个时候，有新的试剂来控制人类和动物的危险病原菌是至关重要的。

RNA polymerase is a fundamental cellular machinery responsible for converting genetic information stored in DNA to another genetic molecule, called RNA, that can then be converted to protein or act in another regulatory capacity. Accessing information in DNA occurs in a complex, highly controlled process called gene transcription and the core molecular machinery, the RNAP enzyme, is conserved from bacteria to humans. Gene transcription is a highly regulated event in development and a major response to growth and environmental stimuli in all known living systems. Although significant advance has been made towards understanding how RNAP functions as an enzyme, including the work recognised by the Nobel Prize in Chemistry in 2006, how RNAP itself is controlled by factors that signal special cellular states and events, is still poorly understood. We use the bacterial RNAP and its major variant sigma factor, sigma54, as a simplified model system to study how RNAP stays in an inhibited state and how activator proteins, acting remotely from where transcription will start, utilise cellular energy in the form of a high energy molecule called ATP, to convert the RNAP from an inactive enzyme to a transcriptionally competent enzyme. We have just determined the crystal structure of RNAP-sigma54 so that we now have a detailed view of what RNAP-sigma54 looks like. Our structure explains how sigma54 maintains RNAP in an inhibited state. Furthermore, we discovered many of the inhibitory strategies we see are shared to some extent by other bacterial and eukaryotic factors and reveal there are conserved hotspots in RNAP that are targeted to varying degrees by different elements and transcriptional factors to fine-tune transcription inhibition. In this current proposed research, we plan to utilise our newly acquired knowledge, experience and reagents already generated to address fundamental questions on how this inhibited state is relieved by activator. This is likely to shed light into how RNAP in humans, plants and animals is activated. Furthermore, we want to exploit the structural features of the inhibited state to design novel antibiotics that inhibit gene transcription by attacking important sites and surfaces on the bacterial RNAP enzyme that have not been targeted before for drug therapies. Inhibiting bacterial RNAP, and hence gene transcription, is a validate antibiotic strategy e.g. in controlling TB infections, so our work should provide novel avenues for effective antibiotic development at a time when it is crucial to have new reagents to control dangerous pathogenic bacteria of humans and animals.