结核分枝杆菌“GG”启动子新元件鉴定及参与转录起始的分子机制研究

Promoter is a regulatory DNA region recognized by RNA polymerase in initiating gene transcription. The classical prokaryotic promoters contain “-10”, “-35” or “TGN” elements. Generally, a promoter containing two of these elements can be successfully recognized by RNA polymerase. However, most of the promoters in Mycobacterium tuberculosis (Mtb) have been shown to contain only one of these characterized elements, “-10”. How do these promoters be recognized by RNA polymerase and initiate transcription remain obscure. In our previous work, we have identified a new type of promoter in Mtb, whose activity is strictly dependent on both the “-10” element as well as an uncharacterized “GG” element upstream of this “-10” region. In the current project, we will systematically characterize the roles and mechanisms of this “GG” element in transcription initiation. For this purpose, we will apply genetic and biochemical approaches, e.g., mutational analyses, in vitro transcription assays, bacterial gene knock-out and complementation tests, and reporter gene fusion, to examine the roles of this “GG” element in activating promoter activities both in vitro and in vivo. Furthermore, the details of how bacterial RNA polymerases recognize this “GG” element and which transcriptional processes this element contributes to will also be characterized. The genome-wide distribution of this “GG” element in Mtb will be further analyzed globally by both bioinformatics prediction and in vitro transcriptome based promoter identification. With all these investigations, we aim to characterize a new promoter element in Mtb and reveal a novel mechanism for transcription initiation, which would broaden our knowledge on mycobacterial promoter and transcription.

细菌启动子经典元件包括“-10”、“-35”和“TGN”等，一般当启动子包含其中两种以上元件时即可被RNA聚合酶识别并起始转录；但结核分枝杆菌（Mtb）中大部分启动子目前只发现典型的“-10”元件，它们如何被RNA聚合酶识别起始转录尚不清楚。申请人前期在Mtb中发现了一类特殊的启动子，其活性依赖于“-10”区及其上游一从未报道过的“GG”元件。本项目将围绕该“GG”启动子新元件在转录起始过程中的功能和作用机制开展研究，拟采用启动子突变、体外转录、基因敲除及回补、报告基因融合等方法，在体内和体外系统分析“GG”新元件对Mtb启动子转录活性的影响、参与的转录起始步骤、被RNA聚合酶识别的机制及在基因组中的分布和功能等。通过对“GG”启动子新元件的深入研究，本项目预期将在Mtb中揭示一种新的转录起始机制，为更全面认识Mtb启动子和转录等基础生物学问题提供新的科学依据。

启动子识别是转录起始过程非常重要的步骤。细菌启动子经典元件包括保守的“-10”和“-35”元件，但结核分枝杆菌(Mtb)等一些细菌中大部分启动子目前只发现典型的“-10”元件，它们如何被RNA聚合酶识别起始转录尚不清楚。在本项目的支持下，我们系统探究了一种新类型的启动子，取得了以下主要结果：. 1.“G-14G-13”启动子新元件鉴定。通过分枝杆菌启动子序列分析，我们发现70%以上的启动子-10元件上游-13或-14位置至少存在一个G核苷酸。通过对不同启动子的“G-14G-13”进行突变，我们发现 “G-14G-13”序列在Mtb中是一个广泛存在的启动子激活序列，我们将其命名为“G-14G-13”启动子新元件。. 2.“G-14G-13”启动子元件识别机制研究。为探究Mtb中“G-14G-13”新元件在转录起始过程中的功能，我们首先通过体外转录和生化分析证明了“G-14G-13”新元件直接参与转录起始过程。随后，我们构建σB不同位点的突变，发现169位谷氨酸是识别新元件的关键位点，并从结构上解释了该位点与“G-14G-13”互作的机制。. 3.“G-14G-13”新元件启动子在细菌压力耐受过程的功能。我们检测了Mtb不同启动子在生长周期内启动子活性的变化。发现当分枝杆菌进入稳定生长期，经典的启动子活性呈下降趋势，而包含“G-14G-13”元件的启动子活性则无明显变化。由此，我们提出“G-14G-13”元件可能参与细菌压力耐受过程。基于此，我们验证了“G-14G-13”元件在耐酸、异烟肼耐受等压力耐受过程中的功能。. 项目执行期间已发表标注该基金资助的论文5篇。本项目在Mtb等细菌中揭示一种新的转录起始机制，为更全面认识细菌启动子和转录过程等基础生物学问题提供新的科学依据。