Quantitative Studies of the Biotin Regulatory System

所有生物体中遗传信息的转录必须响应细胞外和细胞内信号，例如代谢产物水平、细胞-细胞相互作用和配体对细胞表面受体的占据。多功能转录调节蛋白提供了一种将这些信号直接连接到转录起始过程的有效手段。这些转录因子的定量研究将揭示分子开关机制，使密切耦合的一系列生理信号的遗传调控。在大肠杆菌中，营养素生物素的产生通过多功能转录因子BirA与细胞需求相关联。BirA是一种变构的DMA结合蛋白，并且是催化翻译后 将生物素添加到羧化酶中。生物素转移反应中的中间体bio-5 '-AMP， 还通过显著增强BirA同源二聚化的能量来激活蛋白质的位点特异性DNA结合。因此，生物-5 '-AMP结合阻遏物单体的结构和动力学性质的结果的阐明将提供对变构活化途径的深入了解。 这些研究将采用停流荧光测量效应器结合和结构分析的复合物，使用H/D交换耦合到质谱。假设BirA从转录阻遏物到酶的功能转换是通过蛋白质伴侣的转换发生的。此外，由于阻遏物的相同表面被提议用于两种蛋白质：蛋白质相互作用，预期分子模拟有助于切换过程。热力学和动力学测量的替代蛋白质：蛋白质的相互作用将揭示使用一个单一的表面的替代相互作用的分子基础，这是中央的转录开关。表面等离子体共振，稳态和停流荧光和猝灭流方法将用于这些测量。将使用X射线晶体学对相关复合物进行结构分析。

Transcription of genetic information in all organisms must be responsive to extracellular and intracellular signals such as levels of metabolytes, cell-cell interactions, and occupancy of cell surface receptors by ligands. Multifunctional transcription regulatory proteins provide an efficient means of directly linking these signals to the transcription initiation process. Quantitative studies of these transcription factors will reveal the molecular switching mechanisms that enable intimate coupling of an array of physiological signals to genetic regulation. In E.coli, production of the nutrient, biotin, is linked to cellular demand by BirA, a multi-functional transcription factor. BirA is an allosteric DMA binding protein and the enzyme that catalyzes posttranslational addition of biotin to a carboxylase. The intermediate in the biotin transfer reaction, bio-5'-AMP, also activates the protein for site-specific DNA binding by dramatically enhancing the energetics of BirA homodimerization. Thus, elucidation of the consequences of bio-5'-AMP binding for the structural and dynamic properties of the represser monomer will provide insight into the pathway of allosteric activation. These studies will employ stopped-flow fluorescence measurements of effector binding and structural analysis of the complexes using H/D exchange coupled to mass spectrometry. The functional switch of BirA from transcription represser to enzyme is hypothesized to occur via switching of protein partners. Moreover, as the same surface of the represser is proposed to be utilized for the two protein:protein interactions, molecular mimicry is anticipated to contribute to the switching process. Thermodynamic and kinetic measurements of the alternative protein:protein interactions will reveal the molecular basis of the use of a single surface for the alternative interactions that are central to this transcriptional switch. Surface Plasmon Resonance, steady-state and stopped-flow fluorescence and quench-flow methods will be used for these measurements. Structural analysis of the relevant complexes will be carried out using x-ray crystallography.