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

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。

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.