Abbott J, Beckett D
Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore 21228.
Biochemistry. 1993 Sep 21;32(37):9649-56. doi: 10.1021/bi00088a017.
Regulation of biotin biosynthesis and retention in Escherichia coli depends on a complex set of coupled protein-protein, protein-nucleic acid, and protein-small molecule interactions. The complexity of the biotin system is analogous to that found in gene regulatory systems from other prokaryotes and from eukaryotes. Quantitative understanding of these systems requires thermodynamic studies of the individual contributing interactions. We have initiated such studies of the biotin regulatory interactions. The assembly states of the biotin operon repressor (BirA) and its complex with the allosteric effector, bio-5'-AMP, have been determined by analytical gel filtration chromatography. Both the apo- and holo-repressors are monomeric at protein concentrations several orders of magnitude higher than those required for DNA binding. Results of stoichiometric DNA binding measurements indicate that the BirA-biotin operator (bioO) complex consists of two holo-repressor monomers per operator site. Equilibrium binding of BirA to bioO has been measured using the quantitative DNase footprint technique. Analysis of the data indicates that the binding process is best described by a cooperative model. An upper limit for the cooperative free energy is estimated to be between -2.0 and -3.0 kcal/mol.
大肠杆菌中生物素生物合成与保留的调控取决于一系列复杂的蛋白质-蛋白质、蛋白质-核酸和蛋白质-小分子相互作用。生物素系统的复杂性类似于其他原核生物和真核生物基因调控系统中的复杂性。对这些系统的定量理解需要对各个相互作用进行热力学研究。我们已经开始对生物素调控相互作用进行此类研究。通过分析凝胶过滤色谱法确定了生物素操纵子阻遏物（BirA）及其与变构效应物bio-5'-AMP的复合物的组装状态。在比DNA结合所需浓度高几个数量级的蛋白质浓度下，脱辅基和全阻遏物均为单体。化学计量DNA结合测量结果表明，BirA-生物素操纵基因（bioO）复合物每个操纵基因位点由两个全阻遏物单体组成。使用定量DNase足迹技术测量了BirA与bioO的平衡结合。数据分析表明，结合过程最好用协同模型来描述。协同自由能的上限估计在-2.0至-3.0千卡/摩尔之间。
