Structure/function analyses of essential mycobacterial transcription regulators

 描述（由申请人提供）：结核分枝杆菌（Mtb）导致致命的传染病结核病，结核病继续困扰着世界人口，估计世界上有三分之一的人受到感染。CardD和RbpA最近被鉴定为直接RNA聚合酶（RNAP）结合蛋白，其是病原体Mtb中转录的必需调节剂。CardD广泛分布于细菌中，包括分枝杆菌和栖热菌属。我们的初步结果表明，CardD利用一个独特的分子机制来调节转录。 RbpA是仅在放线菌家族的细菌中发现的RNAP的调节剂，包括天蓝色链霉菌（Sco）和Mtb。像CardD一样，RbpA与其他转录调节因子没有相似性，是分枝杆菌生长所必需的，也正调节rRNA转录。 CardD和RpbA都是不寻常的转录调节因子，它们不按照细菌转录激活因子的标准范式发挥作用。我们提出了体外结构和生化方法的组合，并在体内的方法来阐明转录调控的分子机制，由CardD和RbpA。 初步结果包括含CardD的Thermus转录起始复合物的晶体结构（4 μ m分辨率）。我们建议改善这些结构的结果，使用生物化学和生物物理学的方法来测试这些结构所产生的假设，以及确认Mtb卡通过相同的机制作为Thermus卡的功能。 我们还获得了与其RNAP靶之一Mtb sA结合的Mtb RbpA的2.2 nm分辨率晶体结构。我们提出了生物化学和生物物理的方法来测试假设，从这种结构和映射与分枝杆菌RNAP的额外的相互作用。最后，我们建议结晶和确定分枝杆菌RNAP转录起始复合物的结构。

 DESCRIPTION (provided by applicant): Mycobacteria tuberculosis (Mtb) causes the deadly infectious disease, tuberculosis, which continues to plague the world's population, with an estimated one third of the world infected. CarD and RbpA have recently been identified as direct RNA polymerase (RNAP) binding proteins that are essential regulators of transcription in the pathogen Mtb. CarD is widely distributed among bacteria, including Mycobacterium and Thermus species. Our preliminary results indicate that CarD utilizes a distinct molecular mechanism for regulating transcription. RbpA is a regulator of RNAP found only in the actinomycete family of bacteria, including Streptomyces coelicolor (Sco) and Mtb. Like CarD, RbpA has no similarity to other transcription regulators, is essential for growth in mycobacteria and also positively regulates rRNA transcription. Both CarD and RpbA are unusual transcription regulators that do not function according to the standard paradigm for bacterial transcription activators. We propose a combination of in vitro structural and biochemical approaches, and in vivo approaches to elucidate the molecular mechanisms for transcription regulation by CarD and RbpA. Preliminary results include crystal structures of thermus transcription initiation complexes containing CarD (4 Å-resolution). We propose to improve on these structural results, use biochemical and biophysical approaches to test hypotheses that arise from these structures as well as to confirm that Mtb CarD functions through the same mechanism as thermus CarD. We have also obtained a 2.2 Å-resolution crystal structure of Mtb RbpA bound to one of its RNAP targets, the Mtb sA. We propose biochemical and biophysical approaches to test hypotheses that arise from this structure and to map additional interactions with the mycobacterial RNAP. Finally, we propose to crystallize and determine the structure of mycobacterial RNAP transcription initiation complexes.