THERMODYNAMICS & MECHANISMS OF PROTEIN/DNA INTERACTIONS

我们的长期目标是建立一般的热力学和动力学- 控制大分子识别和蛋白质的机械原理- 核酸相互作用（PNAI）在水溶液中，并应用这些 将结构与功能联系起来。我们建议获得一个定量的 了解参与调控的关键位点特异性PNAI的功能 E.大肠杆菌（Lac阻遏子-lac操纵子，RNA 聚合酶启动子），其将延伸到其他原核和真核基因 控制正常和异常细胞过程的调节蛋白 发展 A）DNA中偶联构象变化的热力学特征 (e.g.扭结、平滑弯曲、熔化）和蛋白质中的（例如折叠， 将在PNAI中推导并用于解释 热力学（delta-C-obs、TS、TH、SK-obs）， 构象变化 B）Lacl头盔（HP）和Lacl核心的贡献 将剖析四聚体与操纵子的结合。delta-C-obs，TS， TH和SK-obs将被确定为野生型和变体的相互作用。 Lacl HP与操作员通过量热法和沉降平衡法进行测试 我们的假设，即HP在没有DNA的情况下部分非结构化， 折叠成独特的结构。四聚体-算子平衡将 通过作为侧翼DNA长度的函数的过滤器结合来研究 来测试我们的提议，即Lacl核心和 侧翼非操纵基因DNA（通过缠绕和成环）稳定1：1 络合物，并且在低盐下竞争性地使2：1络合物不稳定。 C）反应的机理途径、中间体和瓶颈动力学步骤， “封闭”络合物异构化为功能性“开放”络合物， APR启动子将使用来自E.大肠杆菌（E- σ 70）和嗜热真细菌。热力学、动力学和 足迹研究将被用来测试我们的建议，关闭 聚合酶的钳口和打开启动子的初始阶段发生 是动力学的瓶颈中间型开环启动子 配合物将被表征，以研究Mg 2+和序列的作用 打开转录起始点的步骤。荧光研究 σ（70）与核心聚合酶结合的热力学和动力学 将测试的建议，结合西格玛（70）打开钳口的核心 并揭示了sigma的DNA识别结构域（70）。

Our long term goals are to establish general thermodynamic and kinetic- mechanistic principles which govern macromolecular recognition and protein- nucleic acid interactions (PNAI) in aqueous solution, and to apply these in relating structure to function. We propose to obtain a quantitative understanding of function of key site-specific PNAI involved in regulation of transcription initiation in E. coli (Lac repressor-lac operator, RNA polymerase-promoter), which will extend to other pro- and eukaryotic gene regulatory proteins which control the processes of normal and abnormal cell development. A) The thermodynamic signatures of coupled conformational changes in DNA (e.g. kinking, smooth bending, melting) and in the protein (e.g. folding, hinge-bending) in PNAI will be deduced and used to interpret the thermodynamics (delta-C-obs, TS, TH, SK-obs) of interactions involving conformational changes. B) Contributions of the Lacl headpiece (HP) and the core of the Lacl tetramer to operator binding will be dissected. Values of delta-C-obs, TS, TH and SK-obs will be determined for interactions of wildtype and variant Lacl HP with operator by calorimetry and sedimentation equilibrium to test our hypothesis that HP is partially unstructured in the absence of DNA, and folds to a unique structure upon binding. Tetramer-operator equilibria will be investigated by filter binding as a function of length of flanking DNA to test our proposals that coulombic interactions between the Lacl core and flanking nonoperator DNA (via wrapping and looping) stabilize 1:1 complexes, and competitively destabilize the 2:1 complex at low salt. C) The mechanistic pathway, intermediates, and bottleneck kinetic step in isomerization of the "closed" complex to the functional "open" complex at the APR promoter will be determined using RNA polymerases from E. coli (E- sigma70) and a thermophilic eubacterium. Thermodynamic, kinetic and footprinting studies will be used to test our proposal that closing the jaws of polymerase and the initial stages of opening the promoter occur together and are the kinetic bottleneck. Intermediate open-promoter complexes will be characterized to study the roles of Mg2+ and the sequence of steps in opening the transcription start site. Fluorescence studies of the thermodynamics and kinetics of binding sigma(70) to core polymerase will test the proposals that binding of sigma(70) opens the jaws of core and unmasks the DNA recognition domain of sigma(70).