Sigma factor networks of M. tuberculosis

描述（由申请人提供）：细菌应激反应的一个关键方面是通过改变sigma因子（一个决定启动子特异性的亚基）来重编程RNA聚合酶（RNAP）。在RNAP全酶中用一个sigma因子代替另一个sigma因子导致迄今为止沉默的细胞功能的表达。这些新表达的功能导致了细菌适应新环境条件和生存所需的代谢、形态和生理变化。人类病原体结核分枝杆菌建立和传播感染的能力与广泛的基因表达重编程密切相关，这种基因表达重编程允许这种微生物在感染期间在复制（生长）和非复制（休眠）状态之间切换。对复杂基因调控的需求反映在专性病原体中，结核分枝杆菌的辅助sigma因子/基因组大小比最高。我们工作的前提是，了解结核分枝杆菌的应激反应需要阐明这种微生物的sigma因子的生物学。sigma因子生物学的核心是转录因子和转录后因子之间的调节相互作用。在目前的R21应用中，我们建议重建sigma因子转录网络的结构，并测试网络对应激条件的响应。实验方法是围绕三个目标建立的，其特点是测试网络的复杂性不断增加。这些包括：(1)确定大肠杆菌中表达的结核分枝杆菌sigma因子之间的直接调节相互作用，(2)评估应激反应无关的sigma因子基因激活对结核分枝杆菌中下游sigma因子基因表达的影响，以及(3)表征暴露于抑菌应激源后的sigma网络反应。成功完成这些目标将使我们能够确定sigma因子之间所有直接和间接的转录相互作用，并为未来重建更大、多尺度的调控网络提供假设，该网络将包括sigma因子活性的转录后控制以及其他类型的压力传感和调控因子的参与。对sigma因子网络的充分了解将有助于识别在生长和休眠之间的发育转换中起作用的sigma因子。这些sigma因子（以及控制其表达和活性的调控途径）成为抗结核干预的潜在靶点。

DESCRIPTION (provided by applicant): A critical aspect of the bacterial stress response is the reprogramming of RNA polymerase (RNAP) by changing the sigma factor, a subunit that determines promoter specificity. Substituting one sigma factor with another in the RNAP holoenzyme results in the expression of hitherto silent cellular functions. These newly expressed functions lead to the metabolic, morphological, and physiological changes that are required for bacterial adaptation to the new environmental conditions and survival. The ability of the human pathogen Mycobacterium tuberculosis to establish and transmit infection is tightly associated with the extensive gene expression reprogramming that allows this microorganism to switch between replicating (growth) and nonreplicating (dormancy) states during infection. The need for complex gene regulation is reflected in the highest accessory sigma factor/genome size ratio seen in M. tuberculosis among obligate pathogens. The premise of our work is that understanding the stress response of M. tuberculosis requires elucidating the biology of this microorganism's sigma factors. Central to sigma factor biology are the regulatory interactions among sigma factors, which are transcriptional and post-transcriptional. In the present R21 application, we propose to reconstruct the architecture of the sigma factor transcriptional network and to test the network response to stress conditions. The experimental approach is built around three aims, which are characterized by the increasing complexity of the network tested. These are: (1) to identify direct regulatory interactions between M. tuberculosis sigma factors expressed in E. coli, (2) to assess the effect of stress-response independent activation of sigma factor genes on the expression of downstream sigma factor genes in M. tuberculosis, and (3) to characterize the sigma network response following exposure to bacteriostatic stressors. Successful completion of the aims will enable us to identify all the direct and indirect transcriptional interactions between sigma factors and to generate hypotheses toward future reconstruction of a larger, multi-scale regulatory network that will include post-transcriptional control of sigma factor activity and the involvement of additional classes of stress-sensing and regulatory factors. Full knowledge of the sigma factor network is expected to identify sigma factors that are part of the developmental switch between growth and dormancy. These sigma factors (and the regulatory pathways controlling their expression and activity) become potential targets for anti-tuberculosis intervention.