Positive control of the primary sigma factor of actinomycetes

细菌具有巨大的经济和社会重要性，我们受益于它们合成有价值的产品（例如抗生素，生物燃料），保存食物和生物转化工业废物的能力，同时面临着与病原菌的持续斗争，这些病原菌进化出逃避当前治疗的方法。为了充分利用细菌用于工业过程，并应对新兴病原体所面临的挑战，我们需要了解细菌如何控制其基因的表达。基因表达始于一种称为RNA聚合酶的酶与位于蛋白质编码序列上游的DNA启动子元件的结合。RNA聚合酶（RNAP）在称为转录的过程中催化基因的RNA拷贝的产生，在大多数情况下，然后在蛋白质的产生中由核糖体翻译。转录起始的频率由调节蛋白控制，其中大多数与启动子区的DNA结合，通常也与RNAP相互作用。RNAP是一种多亚基复合物，由五个亚基的核心酶和一个可分离的第六个亚基组成，该亚基被称为sigma，是启动子结合和DNA解链以显示模板链所必需的。在转录起始后不久，在RNAP从启动子逃逸后，σ亚基解离。细菌通常含有多个σ因子，包括一个主要的σ因子，负责活跃生长细胞中的大部分转录，以及几个替代的σ因子，其具有更专门的作用，重新编程RNAP以识别新的启动子组并打开不同的基因组。我们已经发现了一个非正统的转录因子称为RbpA结合到主要的西格玛因子和刺激转录起始。我们不了解RbpA如何刺激转录，但它不结合DNA，因此其机制与标准的DNA结合激活剂不同。RbpA同系物仅在放线菌家族的细菌中发现，其包括具有工业和医学意义的细菌，最值得注意的是链霉菌属和分枝杆菌属。链霉菌属是大多数临床使用的抗生素和许多化学治疗剂的来源。另一方面，分枝杆菌属包括最重要的全球细菌病原体，M。结核病感染了世界三分之一的人口，每年造成近200万人死亡。该提案旨在发展我们对放线菌中转录起始的理解，并对1）抗生素生物合成基因如何转录和2）抑制分枝杆菌RNAP的新药开发具有重要意义，RNAP是一线TB抗生素利福平的靶标。我们发现RbpA与初级σ因子结合，这表明它在转录起始中起主要作用，这与当该蛋白质不存在于模型链霉菌菌株S中时生长速率的显著减慢一致。天蓝色。在这个项目中，我们将研究如何RbpA影响活动的西格玛通过监测本地化的西格玛染色体DNA在其存在或不存在。我们还将确定RbpA单独的结构和当结合到sigma时的结构，这将使我们能够建立RbpA如何在更大的RNAP复合物的背景下工作的模型，并可能揭示抑制它的新方法。最后，我们将联合收割机的结构模型与新的遗传和生物化学方法相结合，以了解RbpA如何激活转录。该项目的结果将对放线菌中转录如何启动产生广泛的影响，并将引起其他有兴趣寻找抑制RNA聚合酶新方法的研究人员的兴趣。

Bacteria are of enormous economic and social importance and we benefit from their ability to synthesise valuable products (e.g. antibiotics, biofuels), preserve food, and bio-transform industrial waste, while facing a constant battle against pathogenic bacteria that evolve ways of evading current treatments. In order to fully exploit bacteria for industrial processes and to meet challenges faced by emerging pathogens we need to understand how bacteria control the expression of their genes. Gene expression starts with the binding of an enzyme called RNA polymerase to DNA promoter elements that are located upstream of protein coding sequences. RNA polymerase (RNAP) catalyses the production of an RNA copy of the gene in a process called transcription which, in most cases, is then translated by ribosomes in the production of proteins. The frequency of transcription initiation is controlled by regulatory proteins, most of which bind to DNA in the promoter region, often also interacting with RNAP. RNAP is a multi-subunit complex consisting of a core enzyme of five subunits and a dissociable sixth subunit called sigma that is required for promoter binding and the melting of DNA to reveal the template strand. Soon after transcription initiation, after RNAP has escaped from the promoter, the sigma subunit dissociates. Bacteria usually contain multiple sigma factors including one essential primary sigma factor responsible for most transcription in actively growing cells, and several alternative sigma factors with more specialised roles that reprogrammed RNAP to recognise new sets of promoters and switch on distinct groups of genes. We have discovered an unorthodox transcription factor called RbpA that binds to the primary sigma factor and stimulates transcription initiation. We do not understand how RbpA stimulates transcription, but it does not bind DNA and so its mechanism is distinct from standard DNA-binding activators. RbpA homologues are only found in the actinomycete family of bacteria, which includes bacteria of industrial and medical significance, most notably the Streptomyces and Mycobacteria genera. The Streptomyces genus is the source of most clinically-used antibiotics and many chemotherapeutic agents. On the other hand the Mycobacterium genus includes the most important global bacterial pathogen, M. tuberculosis, which infects a third of the world's population and kills approaching 2 million humans per year. This proposal aims to develop our understanding of transcription initiation in actinomycetes and has important implications for 1) how antibiotic biosynthetic genes are transcribed and 2) the development of new drugs to inhibit mycobacterial RNAP, the target of the front-line TB antibiotic rifampicin. Our finding that RbpA binds to the primary sigma factor suggests that it plays a major role in transcription initiation, which is consistent with a dramatic slowing of growth rate when the protein is absent from the model Streptomyces strain S. coelicolor. In this project we will investigate how RbpA influences the activity of sigma by monitoring the localisation of sigma on chromosomal DNA in its presence or in its absence. We will also determine the structure of RbpA alone and when bound to sigma, which will allow us to build a model for how RbpA works in the context of the larger RNAP complex and might reveal new ways to inhibit it. Finally we will combine the structural models with new genetic and biochemical approaches to understand how RbpA activates transcription. The outcomes of the project will have wide ranging implications for how transcription initiates in actinomycetes and will be of interest to other researchers that are interested in finding new ways to inhibit RNA polymerase.