Kinetic regulation of mycobacterial transcription

项目概要/摘要 所有细菌中的转录都是通过单核RNA聚合酶（RNAP）实现的，RNAP与 α-亚基形成RNAP全酶，结合DNA启动子序列，并启动转录。最 转录调节发生在起始水平，转录因子通过以下方式介导这种调节： 直接调节RNAP和启动子之间的相互作用， 在封闭和开放RNAP-启动子复合物（分别为RPc和RPo）之间，或影响 启动子逃逸。我们最近发现，结核分枝杆菌（Mtb）中的转录显著降低， 与模式菌E. Mtb RNAP形成固有不稳定的RPo复合物， 与E. coli RNAP。此外，结核分枝杆菌具有两个必需的转录因子，CardD和RbpA， 不存在于E.杆菌我们以前已经表明，这些因素稳定分枝杆菌RPo，虽然通过 不同的机制，并能够协同和显着改变Mtb RNAP RPo的动力学 形成，使其反映了那些E。coli RNAP。我们一直在体内研究CardD和RbpA的活性 和体外，并提出了动力学机制，为每个因素的背景下，管家σA RNAP 核糖体RNA（rRNA）rrnAP 3启动子上的全酶。我们的研究有助于 了解这些基本转录因子的基本特性，并揭示了对 结核分枝杆菌转录的独特性质。然而，CardD和RbpA活动仅在少数几个国家进行了检查 分枝杆菌启动子的序列元件与在E.大肠杆菌，尽管一般来说 Mtb启动子与E.杆菌此外，CardD仅在以下背景下进行了研究： σA RNAP全酶，尽管调节细菌的12 Mtb替代σ因子的重要性 发病过程中遇到的应激反应。因此，我们仍然不知道CardD和RbpA如何影响 Mtb染色体内绝大多数基因的表达以及这种调节如何有助于 在感染期间Mtb经历的条件下的存活力。在这个项目中，我们将测量结核分枝杆菌的动力学 使用快速混合停流技术以及高分辨率单分子方法引发， 解决如何CardD和RbpA差异影响基因表达从不同的启动子，并在上下文中， 在感染期间，细菌应激反应所必需的替代全酶。我们的目标是：（1） 验证CarD和RbpA可以根据启动子激活或抑制转录的假设 上下文，（2）扩展我们的因子依赖性分枝杆菌转录起始的动力学模型，和（3） 确定CardD和RbpA如何受到替代sigma因子的影响。这些目标的实现将导致 从全基因组Mtb转录调控的整体观点来看， 超越了传统的模型系统。这些研究也将提供深入了解结核病的机制 发病机制，可能会告知未来的治疗策略的发展。

PROJECT SUMMARY/ABSTRACT Transcription in all bacteria is achieved by a single core RNA polymerase (RNAP) which associates with a σ-subunit to form an RNAP holoenzyme, bind DNA promoter sequences, and initiate transcription. Most transcriptional regulation occurs at the level of initiation and transcription factors mediate this regulation by directly modulating the interaction between RNAP and the promoter, manipulating the rates of interconversion between closed and open RNAP-promoter complexes (RPc and RPo respectively), or affecting the rate of promoter escape. We have recently shown that transcription in Mycobacterium tuberculosis (Mtb) is considerably different from that in the model bacterium E. coli in that Mtb RNAP forms inherently unstable RPo complexes as compared to E. coli RNAP. Furthermore, Mtb possess two essential transcription factors, CarD and RbpA, that are absent from E. coli. We have previously shown that these factors stabilize mycobacterial RPo, albeit through different mechanisms, and are able to cooperatively and dramatically change the kinetics of Mtb RNAP RPo formation such that it mirrors those of E. coli RNAP. We have been studying CarD and RbpA activities in vivo and in vitro and have proposed kinetic mechanisms for each factor in the context of the housekeeping σA RNAP holoenzyme on the ribosomal RNA (rRNA) rrnAP3 promoter. Our studies have been instrumental in understanding the fundamental properties of these essential transcription factors and have revealed insight into unique properties of Mtb transcription. However, CarD and RbpA activities have only been examined on a handful of mycobacterial promoters with sequence elements similar to promoters found in E. coli even though in general Mtb promoters differ considerably from those in E. coli. In addition, CarD has only been studied in the context of the σA RNAP holoenzyme, despite the importance of the 12 Mtb alternative σ-factors that regulate the bacterias response to stresses encounter during pathogenesis. Thus, we still do not know how CarD and RbpA affect expression of the vast majority of genes within the Mtb chromosome and how this regulation contributes to viability under the conditions Mtb experiences during infection. In this project, we will measure the kinetics of Mtb initiation using rapid-mixing stopped-flow techniques as well as high-resolution single-molecule approaches to address how CarD and RbpA differentially affect gene expression from diverse promoters and in the context of alternative holoenzymes essential for bacterial stress responses enacted during infection. Our Aims are to: (1) Test the hypothesis that CarD and RbpA can either activate or repress transcription depending on promoter context, (2) Expand our kinetic model of factor-dependent mycobacterial transcription initiation, and (3) Determine how CarD and RbpA are influenced by alternative sigma-factors. Completion of these Aims will result in a holistic view of Mtb transcriptional regulation genome-wide and will expand paradigms of prokaryotic transcription beyond traditional model systems. These studies will also provide insight into mechanisms of Mtb pathogenesis that may inform the development of future therapeutic strategies.