The Assembly Mechanisms of TBP-Nucleated Complexes

TBP有核配合物的组装机制

• 批准号: 7117667
• 负责人: Lawrence JOHN Parkhurst
• 金额: $ 28.23万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7117667
• 关键词: DNA directed RNA polymerase; Saccharomyces cerevisiae; biophysics; chemical kinetics; chemical stability; circular dichroism; conformation; fluorescence resonance energy transfer; fluorimetry; fungal proteins; gene mutation; genetic promoter element; genetic regulation; genetic transcription; molecular assembly /self assembly; molecular biology; nucleic acid probes; nucleic acid sequence; phenylalanine; protein binding; protein engineering; protein structure function; thermodynamics; transcription factor

DESCRIPTION (provided by applicant): Protein synthesis in humans and other Eukarya begins with transcription of the encoding gene. The first step in this process is the binding of the TATA Binding Protein (TBP) to the 'TATA box' sequence within the gene promoter. Our hypothesis is that the unusual structural, energetic and kinetic characteristics of TBP-TATA complexes and their influence on the assembly of the higher order complexes are critical to effective initiation and regulation of transcription. Key issues relating to the complexity and specificity of these binary complexes are addressed in this proposal, with important biological implications for the rate of formation of the transcription pre-initiation complex. The TBP-TATA complex nucleates assembly of Iranscription Factors (TF) IIA and lIB. Together with TBP, TFIIA and TFIIB constitute the minimal ensemble of proteins that can specifically recruit the requisite enzyme, RNA polymerase II, to a promoter. They are also targets for regulatory proteins. Abnormalities in these molecular events in the early stages of pre-initiation complex formation have been implicated in myriad human diseases. This link between transcription and disease provides an opportunity for targeted intervention. Despite their key roles, quantitative molecular mechanisms have not been established for the assembly interactions of the higher order complexes incorporating TFIIA and TFIIB. The proposed work will characterize the time-dependent thermodynamic and structural changes that compose these TBP-nucleated interactions, the essential first step in developing a quantitative predictive model for the initiation and regulation of transcription. The proposed correlation of these biophysical parameters with the corresponding in vivo transcription activity will give needed insight into the rules governing the transcriptional process. While most studies to date have utilized TBP from yeast, the proposed research includes a detailed characterization of the behavior of human TBP. These studies apply an ensemble of quantitative biophysical approaches including time-resolved spectroscopies that allow the integration of conformational, thermodynamic and kinetic characteristics into a comprehensive model of the structure, function and biology of TBP-nucteated complexes. The long-term goal of our research program is to understand the physical-chemical mechanisms by which eukaryotic gene transcription is initiated and regulated.

描述（由申请人提供）：人类和其他Eukarya中的蛋白质合成始于编码基因的转录。此过程的第一步是TATA结合蛋白（TBP）与基因启动子内的“ TATA盒”序列的结合。我们的假设是，TBP-TATA复合物的异常结构，能量和动力学特征及其对高阶复合物组装的影响对于有效的启动和转录的调节至关重要。在本提案中解决了与这些二进制复合物的复杂性和特异性有关的关键问题，对转录前发射复合物的形成速率具有重要的生物学意义。 TBP-TATA复合物将IRANSCRIPTION因子（TF）IIA和LIB的组装成核。与TBP一起，TFIIA和TFIIB构成了最小的蛋白质集合，可以专门将必要的酶RNA聚合酶II募集到启动子。它们也是调节蛋白的靶标。这些分子事件的异常在发生前生成复合物形成的早期阶段与无数人类疾病有关。转录与疾病之间的这种联系为有针对性的干预提供了机会。尽管它具有关键作用，但尚未建立定量分子机制，用于融合TFIIA和TFIIB的高阶复合物的组装相互作用。拟议的工作将表征构成这些TBP - 核相互作用的时间依赖性热力学和结构变化，这是开发转录启动和调节的定量预测模型的第一步。这些生物物理参数与相应的体内转录活性的提议相关性将为控制转录过程的规则提供必要的见解。尽管迄今为止的大多数研究都利用了酵母的TBP，但拟议的研究包括对人类TBP行为的详细表征。这些研究采用了定量生物物理方法的集合，包括时间分辨光谱法，允许将构象，热力学和动力学特征整合到TBP核能复合物的结构，功能和生物学的全面模型中。我们的研究计划的长期目标是了解启动和调节真核基因转录的物理化学机制。