BIOPHYSICS AND GENETICS OF TBP-TAF INTERACTION DYNAMICS

基因转录调控在人类健康中起着不可或缺的作用，这一过程的失调被证明会导致严重的病理疾病状态。目前转录领域的大多数研究都集中在了解调控特定基因表达的机制上。因此，基因转录的全球调控机制并没有得到太多的关注。这项计划中的研究将探讨TBP(TATA盒结合蛋白)在基因转录的全球调控中所起的作用。TBP是连接三种DNA依赖的RNA聚合酶(RNAP I、II、III)基因转录的唯一公共亚基。因此，TBP在真核转录中具有独特的中心地位。在这项建议中提出的数据表明，相对于大量称为TAFs(TBP相关因子)的途径特异性TBP结合蛋白，细胞中TBP的数量对于转录是有限的。这一结果与大量已发表的生化和遗传数据一致。因此，详细了解决定特定途径的TBP-TAF分布和相互作用动力学的规则可以在非常基本的水平上为基因转录提供见解。这项资助利用紧密协调的遗传和生物物理方法的组合来直接研究TBP-TAF在体外和体内的相互作用动力学。需要检验的中心假设是“细胞内mRNA和tRNA基因转录的比率由与这两个途径相关的TBP和同源TAFs的开启速率、解离率、相对亲和力和细胞内浓度决定。”为了考察这一过程，将进行以下研究：1.准确地测定酵母细胞内TBP和TAFs的浓度。2.区域定向突变方法将被用于产生编码合理选择的酵母RNA POL II和POL III TAFs亚集的基因的TBP结合突变体。3.采用停流荧光各向异性分析方法研究了TBP-TAF复合体的组装/拆解动力学。4.表达突变TAFs的酵母细胞将受到分子遗传学和体内荧光测量的结合，以证明体外测量的TBP-TAF相互作用动力学的变化，导致TBP-TAF在体内的分布模式和基因转录水平的预期变化。

Modulation of gene transcription plays an integral role in human health and, dysregulation of this process has been shown to lead to serious pathological disease states. The majority of current research in the field of transcription is focused on understanding the mechanisms which regulate expression of specific genes. Consequently, global regulatory mechanisms of gene transcription have not received much attention. The research in this proposal will investigate the role that TBP (TATA-box Binding Protein) plays in the global regulation of gene transcription. TBP is the only common subunit linking gene transcription by all three DNA dependent RNA polymerases (RNAP I,II,III). As such, TBP holds a unique, central position in eukaryotic transcription. Data is presented in this proposal which indicates that the amount of TBP in the cell is limiting for transcription, relative to the large number of pathway specific TBP binding proteins termed TAFs (TBP Associated Factors). This result is consistent with a large body of published biochemical and genetic data. Hence, a detailed understanding of the rules which dictate pathway specific TBP-TAF distribution and interaction dynamics could provide insights into gene transcription at a very fundamental level. This grant utilizes a tightly orchestrated combination of genetic and biophysical methods to directly examine TBP-TAF interaction kinetics both in vitro and in vivo. The central hypothesis to be tested is "That the ratio of mRNA and tRNA gene transcription inside the cell is governed by the on-rates, dissociation rates, relative affinities, and Intracellular concentrations of TBP and the cognate TAFs associated with these two pathways." To examine this process the following lines of investigation will be pursued: 1. The intracellular concentration of TBP and the TAFs in yeast cells will be accurately determined. 2. Region-directed mutagenesis methods will be used to generate TBP- binding mutants of the genes encoding a rationally selected subset of yeast RNA Pol II and Pol III TAFs. 3. Stopped-flow fluorescence anisotropy assays will be performed to examine the assembly/disassembly kinetics of each TBP-TAF complex. 4. Yeast cells expressing mutant TAFs will be subjected to a combination of molecular genetic and in vivo fluorescence measurements to demonstrate that changes in TBP-TAF interaction kinetics measured in vitro, result in the predicted change in both TBP-TAF distribution pattern and gene transcription levels in vivo.