MECHANISMS OF TF11/1 FUNCTION AND REGULATION

描述：本提案的目的是研究体外 TFII-I的性质和体内功能。需要TFII-1 转录起始于许多TATA较少的启动子， 含有引发剂元素。这一因素也被认为提供了 基础和调节转录信号的整合， 发起人。TFII-I含有6个串联重复的HLH基序，其可能介导 不同的蛋白质/蛋白质和蛋白质/DNA相互作用。的目标 该建议包括对第一过渡联邦机构职能的详细分析 采用DNA结合研究、结合位点选择、体外 转录和体外诱变。确定TFII的结构- 我将通过生物化学和生物物理方法，包括高 解析晶体学分析该提案还包括 通过磷酸化对TFII-I的表观调节的研究 在体内和体外通过磷酸肽指纹，在体外 诱变和采用显性失活MAPK。的功能 将在体内（动物体内）分析V β Inr元件处的TFII-I 细胞）使用野生型和突变型TFII-I蛋白以及V β衍生的 启动子构建体。

DESCRIPTION: The goals of this proposal are to investigate the in vitro properties and in vivo function of TFII-I. TFII-1 is required for transcription initiation from many TATA-less promoters that instead contain an initiator element. This factor is also thought to provide for the integration of basal and regulatory transcription signals at these promoters. TFII-I contains 6 tandemly repeated HLH motifs that may mediate different protein/protein and protein/DNA interactions. The objectives of the proposal include detailed analysis of the functions of TFII-I employing DNA binding studies, binding site selection, in vitro transcription and in vitro mutagenesis. Determining the structure of TFII- I will be approached by Biochemical and Biophysical methods including high resolution crystallographic analysis. The proposal also includes investigations into the apparent regulation of TFII-I by phosphorylation in vivo and in vitro by phosphopeptide fingerprinting, in vitro mutagenesis and by employing dominant negatives of MAPK. The function of TFII-I at the V beta Inr element will be analyzed in vivo (in animal cells) using wild type and mutant TFII-I protein as well as V beta derived promoter constructs.