Structure-based functional analysis of RNA Polymerase

研究项目概述基因表达是生命的基本要求，被认为是分子生物学的中心教条。这一教条描述了遗传信息从脱氧核糖核酸(DNA)到核糖核酸(RNA)(转录)，再到多肽链(翻译)的定向流动，这些多肽链经过翻译后修饰形成功能蛋白质。细菌RNA聚合酶(RNAP)是基因表达的中心酶，通过它完成核糖核苷三磷酸(NTPs)的聚合。催化核心酶由细菌中的五个亚基组成，RNAP与其他细胞机制相互作用，如翻译、DNA修复和复制。细菌RNAP也是开发抗菌药物的有效靶点。所有生物体中的RNAp都是多亚单位、多结构域的酶，所有这些结构域的功能还不是很清楚。我们的实验室解决了大肠杆菌多亚基RNAP的结构在DNA损伤处停滞，这突出了一些特定结构域的移动，这可能参与翻译和DNA修复机制的信号传递。本实验室还解决了一个小的单亚基相对的多亚基RNAps的结构，突出了活性中心结构域的结构保守性，但在分子的其余部分却存在分歧，从而引发了基于结构的功能比较的研究。目的和目标本项目的总体目标是开发和表征新的RNAP抑制剂。该项目计划对多亚基RNAP及其相对单亚基进行基于结构的功能分析，以深入了解RNAP、核糖体和DNA修复因子之间的相互作用；RNAP结构的功能保守，其他细胞机制和小分子的转录抑制原理。该项目将应用广泛的生化、微生物学、遗传学和结构生物学技术。我们将使用一系列已知的和新的转录抑制物作为RNAps功能的分子探针。除了对细菌RNAP功能的基本见解外，该项目还可能导致发现抑制剂的新靶点和未来作为抗菌药物开发的新候选分子。KEYWORDSCell生物学：了解细胞的基本属性、结构和功能，以及它如何响应和影响其局部环境结构生物学和生物物理学：了解分子和大分子复合体的原子组织以及细胞和生物系统中这些成分之间的动态和功能关系抗菌素耐药性

Research project summaryGene expression, the basic requirement for life, is considered the central dogma of molecular biology. This dogma describes the directional flow of genetic information from deoxyribonucleic acid (DNA) to ribonucleic acid (RNA) (transcription), to polypeptide chains (translation) that undergo post-translational modification to form functional proteins. Bacterial RNA polymerase (RNAp) is the central enzyme of gene expression whereby it accomplishes the polymerization of ribonucleoside triphosphates (NTPs). The catalytic core enzyme consists of five subunits In bacteria, RNAp, interacts with other cellular machineries, such as translation, DNA repair and replication. Bacterial RNAp is also a potent target for the development of antibacterials. RNAps in all living organisms are multisubunit, multidomain enzymes, and the functions of all the domains are still not well understood. Our laboratory solved structures of multisubunit RNAp from Escherichia coli (E.coli) stalled at a DNA lesion, which highlighted movements of some particular domains, which may be involved in signalling to translation and DNA repair machineries. Our laboratory also solved the structure of a small single-subunit relative of multi-subunit RNAps, which highlighted the structural conservation of domains of the active center, but divergence in the rest of the molecule, thus prompting the investigation of structure-based functional comparisons.Aims and objectivesThe overall aim of the project is to develop and characterize new inhibitors of RNAp. The project proposes to perform structure-based functional analysis of multisubunit RNAp and its single-subunit relative to bring insight into: the cross-talk between RNAp, ribosome, and DNA repair factors; functional conservation of the RNAp structure, principles of transcription inhibition by other cellular machineries and small molecules.The project will apply a wide arsenal of biochemical, microbiological, genetics, and structural biology techniques. We will use a range of known and new inhibitors of transcription as molecular probes of the functions of RNAps. Besides fundamental insights into the functions of bacterial RNAp, the project may lead to the discovery of new targets for inhibitors and novel candidate molecules for future development as antibacterials.KEYWORDSCell biology: understanding the fundamental properties, structure and function of the cell and how it responds to and influences its local environmentStructural biology and biophysics: understanding the atomic organisation of molecules and macromolecular complexes and the dynamic, functional relationships between these components in cells and biological systemsAntimicrobial resistance