Mechanism of gene regulation by CcpA bound to cre sites located upstream, downstream and overlapping the regulated promoter

全局转录因子CcpA通过与cre序列结合介导枯草芽孢杆菌中100多个基因的激活或抑制。Cre可以在上游、下游，甚至在调节基因的翻译区中，或与调节启动子重叠。我们建议在体内交联的蛋白质CcpA-Strep结合到启动子代表这些不同的位置的cre元素，然后通过质谱鉴定。利用细菌双杂交分析和表面等离子体共振技术，我们将鉴定与CcpA直接相互作用的蛋白质，并定量它们与CcpA的相互作用在分子动力学和动力学上。我们还将阐明CcpA的哪些辅因子参与了这些相互作用。与CcpA间接相互作用的蛋白质将在泰特控制下以各种水平表达，以阐明它们参与CcpA介导的调节。此外，我们将确定结合到CcpA在葡萄糖独立调节启动子的蛋白质。最有趣的蛋白质-CcpA复合物的晶体结构将被确定。我们希望阐明新的机制的细节CcpA如何完成从这些不同的网站相对于受监管的启动子的转录调控。预期的结果将与一般的细菌转录调节相关。

The global transcription factor CcpA mediates activation or repression of more than 100 genes in Bacillus subtilis by binding to cre sequences. Cre can be upstream, downstream, even as far as in the translated region of the regulated gene, or overlapping the regulated promoter. We propose in vivo crosslinking of proteins to CcpA-Strep bound to promoters representing these different locations of cre elements, followed by their identification by mass spectrometry. Using bacterial two-hybrid analysis and surface plasmon resonance we will identify the directly with CcpA interacting proteins and quantify their interaction with CcpA thermodynamically and kinetically. We will also clarify which cofactors of CcpA are involved in these interactions. Indirectly with CcpA interacting proteins will be expressed at various levels under tet control to elucidate their participation in CcpA mediated regulation. Furthermore, we will identify proteins bound to CcpA at glucose independently regulated promoters. The crystal structures of the most interesting protein-CcpA complexes will be determined. We expect to elucidate novel mechanistic details of how CcpA accomplishes regulation of transcription from such different sites with respect to the regulated promoters. The expected results will be relevant for bacterial regulation of transcription in general.