Organisation and regulation of bacterial enhancer-binding proteins

RNA聚合酶(RNAP)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，然后该分子可以转化为蛋白质或以另一种调节或结构能力发挥作用。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。DNA通常在染色体中组织，染色体将DNA组织成更高的顺序结构。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如何作为一种酶发挥作用方面取得了重大进展，包括2006年诺贝尔化学奖表彰的工作，但它如何受到发出特殊细胞状态和事件信号的因素的控制仍然知之甚少。我们正在研究细菌中一种独特的系统，该系统对细菌应激做出反应，并影响细菌对环境变化的反应能力，从而影响其作为病原体感染或在生物技术环境中繁殖的能力。关键的唯一转录因子，称为sigma54，与RNAP结合，通常抑制RNAP以阻止基因表达。在与利用细胞能量流通的特殊控制蛋白进行一系列复杂的交易后，这个系统随后在重塑事件中被激活，允许RNAP转录关键基因，以响应环境的变化。这些控制激活蛋白对广泛的信号做出反应，并从RNAP远程组织在DNA上。因此，如何将这些成分结合在一起以有效地相互作用，DNA在这个系统中是如何组织的，以及信号是如何调节这个系统的，理解这些都是极其重要的。在目前拟议的研究中，我们计划利用生命科学技术的最新发展，特别是使用电子显微镜，来研究这些复杂的蛋白质-DNA组装，以及它们如何随着环境信号的变化而允许调控基因表达事件。这项工作可能会揭示人类、植物和动物中的RNAP是如何被激活的。此外，我们在转录过程中观察大型复杂组装的方法将使我们更接近在完整染色体和完整细胞的背景下研究这些系统。此外，我们想利用这些高度调控状态的结构特征来设计新的抗生素，以抑制药物治疗的基因转录，因为这个系统虽然对于应对压力很重要，但在一系列条件下对正常的细菌生长并不是必不可少的，但对于许多恶劣环境中的适应来说是重要的，比如宿主。因此，细菌产生抗药性的压力将会较小。这种方法在与其他抗生素联合使用时尤其有效。抑制细菌RNAP，从而抑制基因转录，是一种经过验证的抗生素策略，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 (RNAP) 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 or structural 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. DNA is normally organised in chromosomes which organise DNA into higher order structures. 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 it is controlled by factors that signal special cellular states and events, is still poorly understood. We are studying a unique system in bacteria that responds to bacterial stress and affects the ability of bacteria to respond to environmental changes, therefore affecting its ability to infect as a pathogen or propagate in a biotechnological setting. The key unique transcription factor, called sigma54, binds to RNAP and normally inhibits RNAP to prevent gene expression. Following a set of complex transactions with special control proteins that utilise the energy currency of the cell, a molecule called ATP, this system is then activated in a remodeling event to allow the RNAP to transcribe key genes in response to e.g. changes in the environment. These controlling activator proteins respond to a wide range of signals and are organised remotely on the DNA from RNAP. Therefore how these components are brought together to productively interact with each other and how the DNA is organised in this system as well as how signals regulate this system are extremely important to understand. In this current proposed research, we plan to utilise the latest developments in life sciences technologies, especially using electron microscopy, to study these complex protein-DNA assemblies and how they change upon environmental signals to allow a regulated gene expression event. Such work is likely to shed light onto how RNAP in humans, plants and animals is activated. Furthermore, our approach of looking at large complex assemblies in transcription will bring us one step closer to studying these systems in the context of a complete chromosome and in intact cells. Furthermore, we want to exploit the structural features of these highly regulated states in order to design novel antibiotics that inhibit gene transcription for drug therapies as this system, although important for responding to stress, is not essential for normal bacterial growth under a range of conditions, but is important for many adaptations in hostile environments such as the host. The bacteria therefore will be under less pressure to develop resistance. This approach is especially effective when combined with other antibiotics. Inhibiting bacterial RNAP, and hence gene transcription, is a validated 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.