Regulation of translation initiation by Mycobacterium tuberculosis non-coding RNAs observed at the single-molecule level and in real-time

结核分枝杆菌(Mtb)感染是由单一病原体引起的主要死亡原因。治疗结核分枝杆菌的一个主要问题是其独特的生命周期，具有两种不同的代谢状态：活跃的复制疾病状态和潜伏状态。在后一种状态下，蛋白质合成在全球范围内受到下调，因此细菌对抗生素的敏感性较低。非编码RNA(在细菌中称为小RNA，sRNAs)可以调节这种休眠状态的进入和维持，也与活动性结核病感染有关。SRNAs可以在基因表达的几个阶段发挥作用，并作为基因表达的全球调节因子，但对Mtb sRNAs如何在分子水平上调节Mtb基因表达知之甚少。在这个建议中，我们将建立一个体外多色单分子荧光平台，用于实时跟踪Mtb sRNAs调控的共转录翻译启动，并用体外和体内功能分析验证这些数据。跟踪单个mRNA分子将使我们能够直接看到转录、共转录的mRNA折叠、mRNA与sRNAs的相互作用以及翻译启动是如何实时发生的，并在功能上相互耦合。我们的多色单分子平台和由此产生的机制发现将适用于大量未探索的细菌sRNA。我们这个项目的第一个目标是建立一个单分子荧光显微镜平台，用于实时跟踪MTB共转录翻译的启动。为此，我们将1)建立一种使用Mtb RNA聚合酶实时监测转录延长的方法，2)在体外重建并标记Mtb 30S核糖体亚基，3)建立一种同时实时监测转录延长、mRNA折叠和翻译起始的集成方法，以了解新生mRNA结构如何影响翻译起始动力学。本项目的第二个目标是以MTB 6C SRNA为模型系统，研究Mtb 6C SRNA对翻译启动调控的分子机制。在这里，我们将1)研究6C sRNA的结合动力学如何调节不同mRNA靶标的翻译起始效率，2)研究其他因素如何影响6C sRNA的功能(例如RNA伴侣、反义寡核苷酸)。综上所述，这个单分子平台、建立的方法和发展的概念将使我们能够进一步研究迄今鲜有探索的共转录mRNA结构形成的背景，以及动态RNA结构如何结合细胞信号(sRNA、代谢物、环境条件)将它们转化为特定的细胞输出(例如成功的翻译起始)。RNA是动态和多种多样的，因此很难研究，这项提案将提供应对这些挑战的方法。

Mycobacterium tuberculosis (Mtb) infections are the leading cause of death resulting from a single pathogen. A major problem of treating Mtb is its unique life cycle with two different metabolic states: an active replicating disease state and a latent state. In this latter state, protein synthesis is globally down-regulated and therefore, bacteria are less susceptible to antibiotics. Non-coding RNAs (called small RNAs, sRNAs, in bacteria) may regulate entry into and maintenance of this dormant state and are also involved in active TB infection. sRNAs can act at several stages of gene expression and serve as global regulators of gene expression, but little is known about how Mtb sRNAs work at the molecular level to regulate Mtb gene expression. In this proposal, we will set up an in vitro multi-color single-molecule fluorescence platform for real-time tracking of co-transcriptional translation initiation regulated by Mtb sRNAs and validate these data with in vitro and in vivo functional assays. Tracking single mRNA molecules will allow us to directly see how transcription, co-transcriptional mRNA folding, interactions of the mRNA with sRNAs and translation initiation occur in real-time and are functionally coupled with each other. Our multi-color single-molecule platform and the resulting mechanistic findings will be applicable to a multitude of unexplored bacterial sRNAs. Our first goal with this project is to establish a single-molecule fluorescence microscopy platform for real-time tracking of Mtb co-transcriptional translation initiation. To accomplish this, we will 1) establish an assay for monitoring transcription elongation using Mtb RNA polymerase in real-time, 2) reconstitute and label in vitro Mtb 30S ribosomal subunits and 3) develop an integrated assay for simultaneous real-time monitoring of transcription elongation, mRNA folding and translation initiation to understand how nascent mRNA structure affects translation initiation dynamics. The second goal of this project is to investigate the molecular mechanisms of translation initiation regulation by the Mtb 6C sRNA as a model system. Here we will 1) study how the binding dynamics of the 6C sRNA regulate translation initiation efficiency of different mRNA targets and 2) investigate how other factors affect 6C sRNA function (e.g. RNA chaperones, antisense oligos).In summary, this single-molecule platform, the established methodologies and developed concepts will allow further studies of the so far little explored context of co-transcriptional mRNA structure formation and how dynamic RNA structure incorporates cellular signals (sRNAs, metabolites, environmental conditions) to convert them to a specific cellular output (e.g. successful translation initiation). RNA is dynamic and heterogeneous and therefore difficult to study and this proposal will deliver methodologies to tackle these challenges.