Quantitative Studies of the Biotin Regulatory System

生物体用来响应新陈代谢需求的一个常见机制是通过 特定基因转录的变化。这种反应可以通过复杂的信号发生 转导过程或通过更简单的机制涉及代谢物与 转录因子来调节它们的DNA结合功能。实现以下目标的一个范例 代谢需求与基因表达的沟通是通过直接使用 调节基因转录的代谢酶。在这项建议中，实验被描述为 阐明大肠杆菌生物素蛋白连接酶BIRA对 在分子水平上对生物素的需求。Bira通过催化生物素进入新陈代谢 连接到生物素依赖的羧基酶、乙酰辅酶A羧基酶和结合序列 特异性地结合到生物素操纵子序列以调节生物素生物合成的转录 基因。这种调节回路的功能反映了几种性质，包括变构 由小分子激活，利用单一蛋白质表面进行多个相互作用和动力学 蛋白质功能的控制。在建议的研究中，BIRA的分子机制 将结合生物物理和生化测量来阐明功能开关。 其中包括用等温滴定量热法测量配基结合的平衡 和蛋白质通过沉淀平衡结合，H-D交换的测量耦合到 质谱学和蛋白质结合的动力学测量。合并后的研究将 揭示新陈代谢和细胞间通讯的基本分子和细胞机制 抄写。

A common mechanism utilized by organisms to respond to metabolic demands is through changes in transcription of specific genes. This response can occur through complex signal transduction processes or through simpler mechanisms involving binding of metabolites to transcription factors to regulate their DNA binding function. One paradigm for achieving communication of metabolic requirements to gene expression is through the direct use of metabolic enzymes in regulating gene transcription. In this proposal experiments are described to elucidate the mechanism by which the Escherichia coli biotin protein ligase, BirA, responds to the demand for biotin at the molecular level. BirA funnels biotin into metabolism by catalyzing its linkage to the biotin-dependent carboxylase, acetyl CoA carboxylase, and binds sequence specifically to the biotin operator sequence to regulate transcription of the biotin biosynthetic genes. The function of this regulatory circuit reflects several properties including allosteric activation by a small molecule, use of a single protein surface for multiple interactions and kinetic control of protein function. In the proposed studies the molecular mechanism of the BirA functional switch will be elucidated using combined biophysical and biochemical measurements. These include equilibrium measurements of ligand binding by Isothermal Titration Calorimetry and protein association by sedimentation equilibrium, measurements of H-D exchange coupled to mass spectrometry, and kinetic measurements of protein association. The combined studies will reveal the basic molecular and cellular mechanisms of communication between metabolism and transcription.