结核分枝杆菌感染过程中核糖体基因转录调控研究

Tuberculosis is a chronic and complex disease resulting from infection with the slow-growing pathogen, Mycobacterium tuberculosis, which may be present in intracellular and extracellular forms and is still one of the highest mortality of pathogenic bacteria. Expression of ribosomal genes is every important for its transition between active and dormant state. But the regulation for ribosomal genes has not been well studied. RNA polymerase binding protein CarD was first identified to be involved in the negative regulation of ribosomal genes transcription and involved in M. tuberculosis persistent infection. Recently, another RNA polymerase binding protein RbpA was also shown to be involved in Mycobacterium ribosomal genes regulation. And in difference with CarD, RbpA activates gene transcription. Regarding these, mechanisms for these two regulators in ribosomal genes transcription regulation are still largely unknown. Our project intends to establish an in vitro platform to analysis the regulatory details for CarD and RbpA to M. tuberculosis ribosomal genes transcription. As reported by genome sequencing, there are around 60 ribosomal genes in M. tuberculosis. All targets of ribosomal genes for CarD and RbpA will be identified respectively in our project. Targets in transcription procedures for CarD and RbpA will also be determined to show the mechanism(s) for these two regulators. Interaction sites between RNA polymerase and these two regulators will be exactly examined. For the reason that CarD and RbpA regulate ribosomal genes in opposite way, similarity and difference between CarD and RbpA in regulatory mechanism will be compared throughout these analyses. Taken together, transcription regulation of ribosomal genes in M. tuberculosis by CarD and RbpA will be detailed and mechanism for the transition between active and dormant infection is hope to be revealed. Both CarD and RbpA are specific to bacteria, and are essential in M. tuberculosis, which means both these two protein will be good anti-tuberculosis drug target. Our project is aimed to show the details about the roles of these two proteins, which for sure will promote new anti-TB drug development.

结核分枝杆菌是引起结核病的病原细菌，它感染过程中既可以以胞外形式存在，又可以寄居于胞内以休眠状态长期存在，至今仍是致死率最高的病原细菌之一。结核分枝杆菌核糖体基因的转录水平直接决定其活跃与休眠状态的转换过程，目前有报道RNA聚合酶结合蛋白CarD参与其转录负调控作用，申请人前期工作发现另一个RNA聚合酶结合蛋白RbpA则参与其正调控作用，但目前对二者的调控机理还未有详细探讨。本项目拟建立CarD和RbpA调控结核分枝杆菌核糖体基因转录的体外模型，系统分析二者的调控靶标和调控步骤，并鉴定二者与RNA聚合酶的具体相互作用位点，阐释CarD和RbpA调控核糖体基因转录过程中相互之间的影响及作用机制，最终揭示结核分枝杆菌活跃与休眠状态转换的机制。CarD和RbpA均特异性存在于细菌，在结核分枝杆菌中均是必须蛋白，且调控靶标为细菌生存必须环节，本项目的开展将为抗结核药物开发提供新的靶点。

结核分枝杆菌（Mtb）感染宿主过程中既可以活跃形式又可以休眠状态存在，而其核糖体基因的转录水平直接决定这两种状态的转换过程。研究表明RNA聚合酶结合蛋白CarD 和RbpA参与核糖体基因的转录调控，但具体调控机制还不清楚。本项目针对该问题开展了系统的研究，取得了以下结果：.1. 首次建立了Mtb体外转录分析系统，获得了高纯度的Mtb RNA聚合酶核心酶及σ因子，确定了最佳的体外转录反应体系和反应条件，为研究Mtb转录调控特别是分子机制奠定了基础；.2. 首次构建了Mtb启动子转录起始的σ因子依赖类型鉴定系统，并系统分析了Mtb核糖体基因启动子的分布及起始转录的机制，发现σA可以识别所有检测的核糖体启动子的转录；.3. 系统探讨了CarD和RbpA对σA类型RNA聚合酶及核糖体基因转录的调控作用，发现这两种蛋白均直接激活σA类型RNA聚合酶活性和核糖体基因转录，并鉴定了这两个蛋白的关键功能区域；同时还发现这两种蛋白虽然都可激活转录，但调控转录过程的靶点不同，二者存在协同激活作用。.项目执行期间已发表标注该基金资助的论文3篇。由于CarD 和RbpA均是Mtb的必需蛋白，且调控靶标为Mtb感染状态转换过程的关键蛋白，本项目获得的结果为阐释Mtb的感染转换机制提供了重要基础，也为抗结核药物开发提供新的靶点。