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

CcpA 与位于受调控启动子上游、下游和重叠的 cre 位点结合的基因调控机制

• 批准号: 175812556
• 负责人: Professor Dr. Jörg Stülke, since 10/2010
• 金额: --
• 依托单位: Abteilung für Allgemeine Mikrobiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2010
• 资助国家: 德国
• 起止时间: 2009-12-31 至 2014-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/175812556?language=en
• 关键词: Mechanism; gene; regulation; CcpA; bound

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.

全球转录因子CcpA通过结合cre序列介导枯草芽孢杆菌中100多个基因的激活或抑制。Cre可以在调控基因的上游、下游，甚至远至调控基因的翻译区，也可以与调控启动子重叠。我们建议在体内将蛋白质与CcpA-Strep交联，结合到代表这些cre元件不同位置的启动子上，然后通过质谱法对其进行鉴定。利用细菌双杂交分析和表面等离子体共振技术，我们将直接识别与CcpA相互作用的蛋白，并从热力学和动力学上量化它们与CcpA的相互作用。我们还将阐明CcpA的哪些辅助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控制下以不同水平表达，以阐明它们参与CcpA介导的调节。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复合物的晶体结构将被确定。我们期望阐明新的机制细节，CcpA如何完成从这些不同的位点对受调节启动子的转录调节。预期的结果将与细菌的转录调节有关。

项目成果

Structural and Biochemical Analysis of the Essential Diadenylate Cyclase CdaA from Listeria monocytogenes*

• DOI: 10.1074/jbc.m114.630418
• 发表时间: 2015-01
• 期刊: The Journal of Biological Chemistry
• 作者: J. Rosenberg;A. Dickmanns;P. Neumann;K. Gunka;J. Arens;V. Kaever;J. Stülke;R. Ficner;F. Commichau

The YmdB Phosphodiesterase Is a Global Regulator of Late Adaptive Responses in Bacillus subtilis

• DOI: 10.1128/jb.00826-13
• 发表时间: 2014-01-01
• 期刊: JOURNAL OF BACTERIOLOGY
• 影响因子: 3.2
• 作者: Diethmaier, Christine;Newman, Joseph A.;Stuelke, Joerg
• 通讯作者: Stuelke, Joerg