Structure/function analyses of essential mycobacterial transcription regulators

 描述(申请人提供)：结核分枝杆菌(结核分枝杆菌)引起致命的传染病，结核病，它继续困扰着世界人口，估计有三分之一的世界被感染。最近，CARD和RbpA被确定为直接RNA聚合酶(RNAP)结合蛋白，是病原体Mtb转录的重要调节因子。CARD在细菌中广泛分布，包括分枝杆菌和温热杆菌。我们的初步结果表明，CARD利用了一种不同的分子机制来调节转录。RbpA是一种RNAP的调节子，只在放线菌家族中发现，包括天蓝色链霉菌(Streptomyces coelicolor，Sco)和Mtb。与CARD一样，RbpA与其他转录调控因子没有相似之处，对分枝杆菌的生长是必不可少的，也可以正向调节rRNA转录。CARD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剂的标准范例发挥作用。我们提出了体外结构和生化方法以及体内方法相结合的方法来阐明CARD和RbpA转录调控的分子机制。初步结果包括含CARD的Thermus转录起始复合体的晶体结构(4？分辨率)。我们建议改进这些结构结果，使用生化和生物物理方法来检验由这些结构产生的假说，并确认MTB CARD与Thermus CARD通过相同的机制发挥作用。我们还获得了Mtb RbpA与其RNAP靶之一Mtb Sa结合的2.2？分辨率的晶体结构。我们提出了生化和生物物理的方法来测试从这种结构中产生的假设，并绘制了与分枝杆菌RNAP的额外相互作用图。最后，我们建议结晶和确定分枝杆菌RNAP转录起始复合体的结构。

 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.