Quantitative Studies of the Biotin Regulatory System

在所有生物体中，遗传信息的转录必须响应细胞外和细胞内的信号，如代谢水平、细胞间相互作用和配体对细胞表面受体的占用。多功能转录调控蛋白提供了一种直接将这些信号与转录起始过程联系起来的有效手段。这些转录因子的定量研究将揭示分子开关机制，使一系列生理信号与遗传调控密切耦合。在大肠杆菌中，营养物质生物素的产生与BirA（一种多功能转录因子）的细胞需求有关。BirA是一种变构性DMA结合蛋白和翻译后催化酶

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.