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，该酶与 σ 亚基形成 RNAP 全酶，结合 DNA 启动子序列并启动转录。大多数 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，操纵相互转化的速率 关闭和开放的 RNAP 启动子复合物（分别为 RPc 和 RPo）之间，或影响 启动子逃逸。我们最近表明，结核分枝杆菌 (Mtb) 中的转录水平相当高。 与模型细菌大肠杆菌中的不同之处在于 Mtb RNAP 形成固有不稳定的 RPo 复合物，如 与大肠杆菌 RNAP 相比。此外，Mtb 拥有两个必需的转录因子 CarD 和 RbpA， 大肠杆菌中不存在。我们之前已经证明这些因素可以稳定分枝杆菌 RPo，尽管是通过 不同的机制，并且能够协同并显着改变 Mtb RNAP RPo 的动力学 形成，从而反映了大肠杆菌 RNAP 的形成。我们一直在研究CarD和RbpA的体内活性 和体外，并提出了在管家 σA RNAP 背景下每个因素的动力学机制 核糖体 RNA (rRNA) rrnAP3 启动子上的全酶。我们的研究有助于 了解这些重要转录因子的基本特性，并揭示了 Mtb 转录的独特特性。然而，CarD 和 RbpA 活动仅在少数情况下进行了检查 具有与大肠杆菌中发现的启动子相似的序列元件的分枝杆菌启动子，尽管一般而言 Mtb 启动子与大肠杆菌中的启动子有很大不同。此外，CarD 仅在以下背景下进行了研究： σA RNAP 全酶，尽管调节细菌的 12 个 Mtb 替代 σ 因子很重要 对发病过程中遇到的应激的反应。因此，我们仍然不知道CarD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 Mtb 在感染期间经历的条件下的生存能力。在这个项目中，我们将测量 Mtb 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 CarD 和 RbpA 如何差异性地影响不同启动子的基因表达以及在 感染过程中细菌应激反应所必需的替代全酶。我们的目标是：(1) 检验 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，以及（3） 确定替代西格玛因子如何影响 CarD 和 RbpA。完成这些目标将导致 从全基因组范围内 Mtb 转录调控的整体角度来看，并将扩展原核生物的范式 超越传统模型系统的转录。这些研究还将深入了解 Mtb 的机制 发病机制可能为未来治疗策略的发展提供信息。

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.