STRUCTURAL STUDIES ON THE MASTER REGULATOR OF CARBON CATABOLITE CONTROL IN GRAM

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 碳分解代谢抑制(CCR)是一种全球性的调节机制，使细菌能够优先利用其环境中可用的碳源。在大肠杆菌和其他肠道细菌中，自从J.Monod第一次指出大肠杆菌更喜欢将葡萄糖作为碳源以来，Ccr已经得到了广泛的研究。后来发现，这些细菌中的CCR是通过DNA结合激活蛋白cAMP受体蛋白(CRP)介导的，cAMP受体蛋白在cAMP存在的情况下被诱导与DNA结合。最近的研究表明，既不含CRP也不含cAMP的革兰氏阳性菌使用完全不同的CCR机制。在这些细菌中，CCR的主要调节因子是转录因子，即LacI/GalR家族的碳分解代谢蛋白A(CCPA)。突显其多效性作用的是，该蛋白调节芽孢杆菌中200多个基因，占整个芽孢杆菌基因组的8%以上。作为对高水平1，6-二磷酸果糖的反应，HPR激酶在丝氨酸46处磷酸化含有组氨酸的蛋白(HPR)。SER46磷酸化形式的HPR与CCPA结合，并激活结合其在大CCR操纵子中的Cre DNA位点。因此，磷酸Ser46HPr作为CCPA的辅阻遏子发挥作用，提供了唯一一个激活的LacI/GalR成员通过与另一种蛋白质结合来结合DNA的例子。为了剖析磷酸丝氨酸46Hpr作为辅阻遏子并与CCPA特异性相互作用的分子机制，我们从巨型芽孢杆菌中获得了CCPA-磷酸丝氨酸46HPr-Cre三元位点的晶体。我们还在没有磷酸Ser46Hpr或Cre的情况下生长了CCPA的晶体，以获得CCPA的非激活构象和激活构象。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Carbon Catabolite Repression (CCR) is the global regulatory mechanism enabling bacteria to preferentially utilize the carbon source that is available in their environment. In E. coli and other enteric bacteria, CCR has been extensively studied since J. Monod first noted that E. coli prefer glucose as a carbon source. It was later shown that CCR in these bacteria is mediated through the DNA binding activator protein, cAMP receptor protein (CRP), which is induced to bind DNA in the presence of cAMP. It has more recently been demonstrated that Gram positive bacteria, which contain neither CRP nor cAMP, use an entirely different mechanism of CCR Specifically, in these bacteria, the master regulator of CCR is the transcription factor, the Carbon catabolite protein A (CcpA), a member of the LacI/GalR family. Underscoring its pleiotropic role, this protein regulates over 200 genes in Bacilli, accounting for over 8% of the entire Bacilli genome. In response to high levels of fructose 1,6-bisphosphate, HPr kinase phosphorylates the histidine-containing protein (HPr) at Serine 46. The Ser46 phosphorylated form of HPr binds CcpA and activates to bind its Cre DNA sites in the large CCR operon. Thus, phosphoSer46HPr functions as a corepressor for CcpA, providing the only example of a LacI/GalR member that is activated to bind DNA by binding another protein. To dissect the molecular mechanism whereby phosphoSer46Hpr can function as a corepressor and specifically interact with CcpA, we have obtained crystals of the ternary CcpA-phosphoSer46HPr-Cre site from B. megaterium. We have also grown crystals of CcpA in the absence of phosphoSer46Hpr or the Cre in order to obtain the non-activated as well as the activated conformational state of CcpA.