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- 支配大分子识别和蛋白质的机械原理- 在水溶液中的核酸相互作用(PNAI)，并应用这些 把结构和功能联系起来。我们建议获得一个量化的 对参与调控的关键部位特异性PNAI功能的理解 在大肠杆菌中的转录启动(Lac抑制子-Lac操纵子，RNA 聚合酶启动子)，这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偶联构象变化的热力学特征 (例如，扭曲、平滑弯曲、熔化)和蛋白质中(例如，折叠， 铰链弯曲)将被推导出来并用来解释 相互作用的热力学(增量-C-OBS、TS、TH、SK-OBS) 构象变化。 B)LACL头饰(HP)和LACL核心的贡献 四聚体与操纵子的结合将被剖析。增量-C-OBS、TS、 对于野生型和变异型的相互作用，将确定TH和SK-OBS Lacl HP与操作员通过量热法和沉降法进行平衡测试 我们的假设是，在没有DNA的情况下，幽门螺杆菌是部分无结构的，并且 结合时可折叠成独特的结构。四聚体-算符平衡将 通过过滤结合作为DNA侧翼长度的函数进行研究 来测试我们的建议，即LaCL核和Lacl核之间的库仑相互作用 侧翼非操纵子DNA(通过包裹和环化)稳定在1：1 在低盐条件下，2：1的络合物具有竞争性地不稳定。 C)机械途径、中间体和瓶颈动力学步骤 “封闭”络合物异构化为官能化“开放”络合物 APR启动子将使用来自大肠杆菌(E-R)的RNA聚合酶来确定。 Sigma70)和一株嗜热真细菌。热力学、动力学和 足迹研究将被用来测试我们的建议，即关闭 聚合酶的钳口和启动子打开的初始阶段发生 两者共同构成了动力瓶颈。中间开放启动子 将通过表征络合物来研究镁离子的作用和顺序 打开转录起始站点的步骤。分子的荧光研究 Sigma(70)与核心聚合酶结合的热力学和动力学 将测试西格玛(70)的结合打开核心的下巴的提议 并揭开了Sigma(70)的DNA识别结构域。

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).