基于结构的金黄色葡萄球菌δ亚基依赖型转录分子机制研究

金黄色葡萄球菌日趋严重的耐药性使得新型防治手段的开发迫在眉睫。细菌RNA聚合酶是含5个亚基（2α、β、β'和 ω）的超大复合物，其序列与三维结构在大多数细菌中保守，是重要的药物靶标。金葡菌RNA聚合酶具有一个独特的δ亚基，该亚基直接参与毒力因子表达，缺失δ亚基显著降低菌株黏附、抗免疫细胞清除和宿主体内扩散能力。靶向金葡菌含δ亚基RNA聚合酶可能是控制耐药菌株的一种新颖手段。然而目前金葡菌δ亚基结构、含δ亚基RNA聚合酶复合物结构以及复合物的转录特征和调控机制尚未阐明。我们前期在体外表达、纯化，成功重组了金葡菌含δ亚基RNA聚合酶全酶，在本项目中，我们拟解析金葡菌含δ亚基的转录起始激活、转录延伸暂停、转录终止复合物的三维结构，并从体外和体内深入研究δ亚基在金葡菌转录起始、延伸、终止中发挥的功能。金黄色葡萄球菌这一独特转录分子机器结构的解析，将为靶向该分子机器的抗感染药物设计提供实验基础。

With the increasing antibiotic resistance of pathogenic strain Staphylococcus aureus, the development of new control methods is imminent. Bacterial RNA polymerase is a super-complex consisting of five subunits (2α, β, β' and ω), the sequence and three-dimensional structure of which are highly conserved in most bacteria, but different from human RNA polymerase, therefore bacterial RNA polymerase is an important drug target for new anti-infective drug development. Staphylococcus aureus RNA polymerase has a unique δ subunit, which has been proved to directly participate in the expression of virulence factors genes. The deletion of the δ subunit can significantly reduce the cell adhesion, anti-immune cell clearance and host cell diffusion ability of Staphylococcus aureus strains. Targeting Staphylococcus aureus RNA polymerase with δ subunit may be a novel method to control antibiotic resistant strains. However, the structure of Staphylococcus aureus δ subunit, the structure of Staphylococcus aureus RNA polymerase complexed with δ subunit, and the transcriptional characteristics and regulatory mechanisms of the complexes have not yet been clarified and elucidated.. We have previously expressed, purified and reconstituted the Staphylococcus aureus RNA polymerase holoenzyme containing δ subunit in vitro successfully. In this project, we plan to solve the three-dimensional structure of transcription activation, transcription pause and transcription termination complexes of Staphylococcus aureus, and further characterize the biological functions of the δ subunit in transcriptional initiation, elongation and termination of Staphylococcus aureus in vitro and in vivo. The project will provide the experimental basis for the design of anti-infective drugs targeting this molecular machine by solving this unique transcriptional molecular machine structure of important pathogen S. aureus.