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

全球转录因子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，甚至在受调控基因的翻译区，或者与受调控启动子重叠。我们建议在体内将蛋白质与CCPA-Strep结合到代表这些不同位置的cre元件的启动子上，然后用质谱仪鉴定它们。利用细菌双杂交分析和表面等离子体共振，我们将鉴定与CCPA直接相互作用的蛋白质，并从热力学和动力学上定量它们与CCPA的相互作用。我们还将阐明CCPA的哪些辅因子参与了这些相互作用。间接与CCPA相互作用的蛋白将在Tet调控下在不同水平上表达，以阐明它们参与CCPA介导的调控。此外，我们将在葡萄糖独立调节的启动子上鉴定与CCPA结合的蛋白质。最有趣的蛋白质-CCPA络合物的晶体结构将被确定。我们希望阐明CCPA如何完成针对受调控启动子的不同位点转录调控的新机制细节。预期的结果将与细菌对转录的调控有关。

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.