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。该过程的第一步是将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，TBP-TATA复合物的不同寻常的结构、能量和动力学特征及其对高阶复合物组装的影响对有效的起始和转录调控至关重要。与这些二元复合物的复杂性和特异性相关的关键问题在本提案中得到解决，对转录起始前复合物的形成速度具有重要的生物学意义。TBP-TATA复合物可组装转录因子（TF） IIA和lIB。TFIIA和TFIIB与TBP一起构成了最小的蛋白质集合，可以特异性地将所需的酶RNA聚合酶II募集到启动子上。它们也是调节蛋白的靶标。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。这种转录和疾病之间的联系为有针对性的干预提供了机会。尽管TFIIA和TFIIB具有关键作用，但它们的组装相互作用的定量分子机制尚未建立。这项工作将描述构成这些tbp成核相互作用的时间依赖的热力学和结构变化，这是开发转录起始和调控定量预测模型的重要的第一步。提出的这些生物物理参数与相应的体内转录活性的相关性将为控制转录过程的规则提供必要的见解。虽然迄今为止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行为的详细描述。这些研究应用了定量生物物理方法的集合，包括时间分辨光谱，允许将构象，热力学和动力学特征整合到tbp核配合物的结构，功能和生物学的综合模型中。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节的物理化学机制。

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.