Quantitative Studies of the Biotin Regulatory System

生物体响应代谢需求的一种常见机制是通过 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，涉及代谢物结合到 转录因子调节其DNA结合功能。一种实现 代谢要求与基因表达的沟通是通过直接使用 代谢酶调节基因转录。在该提案中，实验被描述为 阐明了大肠杆菌生物素蛋白连接酶BirA响应于 在分子水平上对生物素的需求。BirA通过催化生物素进入代谢， 连接到生物素依赖性羧化酶，乙酰辅酶A羧化酶，并结合序列 特异性地与生物素操纵子序列结合，以调节生物素生物合成酶的转录。 基因.这种调节回路的功能反映了几种性质，包括变构 通过小分子激活，使用单个蛋白质表面进行多重相互作用和动力学 控制蛋白质功能。在拟议的研究中，BirA的分子机制 功能开关将使用组合的生物物理和生物化学测量来阐明。 这些包括通过等温滴定量热法对配体结合的平衡测量 和蛋白质缔合的沉降平衡，测量H-D交换耦合到 质谱和蛋白质缔合的动力学测量。合并研究将 揭示了代谢和代谢之间通讯的基本分子和细胞机制， 转录。

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.