The Assembly Mechanisms of TBP-Nucleated Complexes

描述（由申请人提供）：人类和其他真核生物的蛋白质合成始于编码基因的转录。该过程的第一步是TATA结合蛋白（TBP）与基因启动子内的“TATA盒”序列结合。我们的假设是，不寻常的结构，能量和动力学特性的TBP-TATA复合物及其对组装的高阶复合物的影响是至关重要的有效启动和转录调控。这些二元复合物的复杂性和特异性有关的关键问题在这个建议中得到解决，具有重要的生物学意义的转录前起始复合物的形成速率。TBP-TATA复合物使转录因子（TF）IIA和IIB的组装成核。与TBP一起，TFIIA和TFIIB构成了可以特异性地将必需的酶RNA聚合酶II募集到启动子的蛋白质的最小集合。它们也是调节蛋白的靶点。在起始前复合物形成的早期阶段，这些分子事件的异常与无数人类疾病有关。转录和疾病之间的这种联系为靶向干预提供了机会。尽管他们的关键作用，定量的分子机制尚未建立更高阶的配合物，将TFIIA和TFIIB的组装相互作用。拟议的工作将表征组成这些TBP核相互作用的时间依赖性热力学和结构变化，这是开发转录起始和调控的定量预测模型的重要第一步。这些生物物理参数与相应的体内转录活性的相关性将提供所需的洞察力的规则，转录过程。虽然迄今为止的大多数研究都使用了来自酵母的TBP，但拟议的研究包括对人类TBP行为的详细表征。这些研究应用了一套定量生物物理方法，包括时间分辨光谱，允许整合的构象，热力学和动力学特性到一个全面的模型的结构，功能和生物学的TBP-nucteated复合物。我们研究计划的长期目标是了解真核生物基因转录启动和调控的物理化学机制。

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.