Kinetic regulation of mycobacterial transcription

项目摘要/摘要 所有细菌中的转录都是通过单核心RNA聚合酶(RNAP)实现的，该聚合酶与 一个σ亚基，形成RNAP全酶，结合DNA启动子序列，启动转录。多数 转录调控发生在起始水平，转录因子通过 直接调节RNAP和启动子之间的相互作用，控制相互转化的速度 在封闭和开放的RNAP-启动子复合体(分别为RPC和RPO)之间，或影响 发起人逃脱了。我们最近发现，结核分枝杆菌(Mtb)的转录水平相当高。 与模式细菌大肠杆菌中的不同之处在于，Mtb RNAP形成固有的不稳定的RPO复合体 与大肠杆菌RNAP相比。此外，结核分枝杆菌还拥有两个必需的转录因子，即CARD和RbpA，它们是 不存在于大肠杆菌中。我们先前已经表明，这些因素稳定了分枝杆菌RPO，尽管是通过 不同的机制，并能够协同并显著改变Mtb RNAP RPO的动力学 形成与E.ColiRNAP相似的结构。我们一直在体内研究CARD和RbpA的活性 并在管家σA RNAP的背景下提出了每个因素的动力学机制 核糖体RNA(RRNA)rrnAP3启动子上的全酶。我们的研究对 了解这些基本转录因子的基本属性，并揭示了 结核分枝杆菌转录的独特性质。然而，CARD和RbpA活动只在少数几个方面进行了检查 具有与在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 MTB启动子与大肠杆菌中的启动子有很大的不同。此外，仅在以下背景下研究了CARD σA RNAP全酶，尽管12Mtb替代σ-调节细菌的因子的重要性 对发病过程中遇到的压力的反应。因此，我们仍然不知道CARD和RbpA是如何影响 结核分枝杆菌染色体中绝大多数基因的表达及其调节作用 在结核分枝杆菌感染期间所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 阐述CARD和RbpA如何在不同启动子的不同背景下影响基因表达 在感染过程中对细菌应激反应至关重要的替代全酶。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)扩展我们的依赖于因子的分枝杆菌转录启动动力学模型，以及(3) 确定卡片和RbpA如何受到备选西格玛因素的影响。这些目标的实现将会带来 从整体上看结核分枝杆菌转录调控的全基因组范围，并将扩大原核生物的范例 超越传统模型系统的转录。这些研究也将有助于深入了解结核分枝杆菌的发病机制。 可能为未来治疗策略的发展提供信息的发病机制。

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.