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中的遗传信息转换为另一种称为RNA的遗传分子，然后该分子可以转换为蛋白质或以另一种调节能力发挥作用。获取DNA中的信息是在一个复杂的、高度受控的过程中进行的，这个过程称为基因转录，而核心分子机制--RNAP酶--从细菌到人类都是保守的。在所有已知的生命系统中，基因转录是一种高度调控的发育事件，是对生长和环境刺激的主要反应。尽管在理解RNAP作为一种酶的功能方面已经取得了重大进展，包括2006年诺贝尔化学奖认可的工作，但RNAP本身是如何由发出特殊细胞状态和事件信号的因素控制的，仍然知之甚少。我们使用细菌RNAP及其主要变体sigma因子sigma54作为一个简化的模型系统，来研究RNAP如何保持在抑制状态，以及激活蛋白如何在转录开始的地方远程作用，以一种名为ATP的高能分子的形式利用细胞能量，将RNAP从失活的酶转化为具有转录活性的酶。我们刚刚确定了RNAP-sigma54的晶体结构，这样我们现在就有了RNAP-sigma54的详细信息。我们的结构解释了sigma54如何将RNAP维持在抑制状态。此外，我们发现我们看到的许多抑制策略在一定程度上与其他细菌和真核因子相同，并揭示了RNAP中存在保守的热点，这些热点被不同的元件和转录因子不同程度地靶向，以微调转录抑制。在目前拟议的研究中，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激活剂如何解除这种抑制状态的基本问题。这可能有助于揭示人类、植物和动物中的RNAP是如何被激活的。此外，我们想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酶上的重要部位和表面来抑制基因转录，这些部位和表面以前没有被作为药物治疗的靶点。抑制细菌RNAP，从而抑制基因转录，是一种有效的抗生素策略，例如在控制结核病感染方面，因此我们的工作应该为有效开发抗生素提供新的途径，而此时控制人类和动物的危险病原体至关重要的是有新的试剂。

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.