The Assembly Mechanisms of TBP-Nucleated Complexes

描述(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。这一过程的第一步是将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络合物独特的结构、能量和动力学特性及其对高阶络合物组装的影响对于转录的有效启动和调控至关重要。本提案讨论了与这些二元复合体的复杂性和特异性有关的关键问题，对转录前起始复合体的形成速度具有重要的生物学意义。TBP-TATA复合体是转录因子(Tf)IIA和Lib的核组装。与TBP一起，TFIIA和TFIIB构成了最小的蛋白质集合，可以特异性地将必要的酶RNA聚合酶II招募到启动子上。它们也是调节蛋白的靶标。在启动前复合体形成的早期阶段，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有针对性的干预提供了机会。尽管TFIIA和TFIIB具有重要的作用，但TFIIA和TFIIB的高阶配合物的组装相互作用的定量分子机制尚未建立。这项拟议的工作将表征组成这些TBP核相互作用的依赖于时间的热力学和结构变化，这是开发转录启动和调控的定量预测模型的关键第一步。所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使人们对转录过程的规则有必要的了解。虽然到目前为止，大多数研究都利用了酵母中的TBP，但拟议的研究包括对人类TBP行为的详细表征。这些研究应用了一系列定量的生物物理方法，包括时间分辨光谱学，允许将构象、热力学和动力学特征整合到TBP-核络合物的结构、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启动和调控真核基因转录的物理化学机制。

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.