Mechanisms of Cooperative Protein-DNA Interactions

蛋白质-DNA 协同相互作用的机制

• 批准号: 6581978
• 负责人: Michael D. Brenowitz
• 金额: $ 37.24万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 2006-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6581978
• 关键词: DNA binding protein; DNA directed RNA polymerase; DNA footprinting; Saccharomyces cerevisiae; X ray crystallography; adenosine triphosphate; biophysics; chemical kinetics; fluorescence spectrometry; fungal genetics; fungal proteins; genetic promoter element; genetic regulation; genetic transcription; hydrolysis; protein protein interaction; protein structure function; sedimentation equilibrium; stoichiometry; stop flow technique; thermodynamics; transcription factor; ultracentrifugation

DESCRIPTION (provided by applicant): The TATA Binding Protein (TBP) plays a central role in the initiation of gene transcription in eukaryotes. In addition, unusual structural features of TBP and its complexes with DNA and regulatory proteins are reflected in equally unusual thermodynamics and kinetics of DNA binding. This concordance of unusual structural and functional properties permits fundamental questions of biophysical chemistry to be addressed in a system of high biological significance. The proposed studies apply both novel and well-established approaches to the study of protein and nucleic acid structure and the interaction of these macromolecules. Specific Aim I seeks to determine whether interdomain interactions within native TATA Binding Protein (TBP) regulate its sequence-specific DNA binding and if so, how. Radiolytic (hydroxyl radical) "protein footprinting" and intrinsic protein fluorescence will be used to map in solution the structures of the nonconserved N-terminal and conserved C-terminal domains of TBP and their interaction. Specific Aim II explores the mechanism(s) do the proteins NC2 and Mot1 positively and negatively regulate S. cerevisiae TBP-promoter interactions. Mot1 is unusual in that it uses the energy derived from ATP hydrolysis to dissociate the long-lived TBP-TATA Box complex from promoters. Radiolytic footprinting, analytical ultracentrifugation and equilibrium and quench-flow DNase I footprinting will be used to explore these cooperative regulatory interactions. Specific Aim III seeks to determine the mechanism by which the protein complex that specifies transcription by RNA Polymerase IIII, TFIIIB (i.e., TBP-TFIIIB70-TFIIIB90), achieve unusual stability when bound to DNA compared to its homolog in the RNA Polymerase II system, TBP-TFIIB. The long-term goal of this research program is to obtain a comprehensive understanding of the physical principles underlying the initiation of gene transcription and its regulation.

描述（由申请人提供）：TATA结合蛋白（TBP）在真核生物中基因转录的开始中起着核心作用。此外，TBP及其与DNA和调节蛋白的复合物的异常结构特征反映在同样异常的热力学和DNA结合的动力学中。这种异常的结构和功能特性的一致性允许在具有高生物学意义的系统中解决生物物理化学的基本问题。拟议的研究将新颖和公认的方法应用于蛋白质和核酸结构的研究以及这些大分子的相互作用。特定目的I旨在确定天然TATA结合蛋白（TBP）中的域间相互作用是否调节其序列特异性DNA结合，如果是，则如何。放射性细胞（羟基自由基）“蛋白足迹”和内在蛋白质荧光将用于在溶液中绘制未经保守的N端和保守的TBP的C末端结构域的结构及其相互作用。特定的目标II探讨了蛋白质NC2和MOT1的机制，对酿酒酵母TBP促进链球菌相互作用进行了负面调节。 MOT1是不寻常的，因为它使用了从ATP水解中得出的能量将长寿命的TBP-TATA盒子与启动子分离。放射性足迹，分析性超速离心，平衡和淬灭DNase I足迹将用于探索这些合作的调节相互作用。具体目的III旨在确定指定RNA聚合酶IIII转录的蛋白质复合物的机制，TFIIIB（即TBP-TFIIIB70-TFIIIB90），与RNA聚合酶II系统中的DNA相比，在与DNA相比，在绑定到DNA时实现了异常稳定性。该研究计划的长期目标是获得对基因转录开始及其调节基础的物理原理的全面理解。