BIOPHYSICS AND GENETICS OF TBP-TAF INTERACTION DYNAMICS

基因转录的调节在人类健康中起着不可或缺的作用，这一过程的失调已被证明会导致严重的病理疾病状态。目前转录领域的大部分研究都集中在了解调节特定基因表达的机制上。因此，基因转录的全局调控机制并没有受到太多的关注。本课题将研究TBP （TATA-box Binding Protein）在基因转录的全局调控中所起的作用。TBP是所有三种DNA依赖性RNA聚合酶（RNAP I，II，III）连接基因转录的唯一共同亚基。因此，TBP在真核生物转录中占有独特的中心地位。该提案中提供的数据表明，与大量被称为TBP相关因子的途径特异性TBP结合蛋白相比，细胞中TBP的数量限制了转录。这一结果与大量已发表的生物化学和遗传数据一致。因此，详细了解特定通路TBP-TAF分布和相互作用动力学的规则可以在非常基础的水平上提供对基因转录的见解。这项资助利用遗传和生物物理方法的紧密结合，直接检查TBP-TAF在体外和体内的相互作用动力学。要验证的中心假设是“细胞内mRNA和tRNA基因转录的比例是由与这两种途径相关的TBP和同源TAFs的on率、解离率、相对亲和力和细胞内浓度决定的。”为了审查这一进程，将进行下列调查：酵母细胞中TBP和TAFs的细胞内浓度将被准确测定。2. 区域定向诱变方法将用于产生编码合理选择的酵母RNA Pol II和Pol III taf子集的基因的TBP结合突变体。3. 将进行停流荧光各向异性分析，以检查每个TBP-TAF复合物的组装/拆卸动力学。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，以证明在体外测量的TBP-TAF相互作用动力学的变化，导致TBP-TAF分布模式和体内基因转录水平的预测变化。

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.