The Role of TFIIB in Transcription Activation

描述(申请人提供)：基因表达主要在转录水平上调节，转录主要在起始水平上调节。适当的基因调控是维持生物体内环境平衡状态的关键事件。这在癌症等疾病中就是例证，在这些疾病中，不适当的基因调控会导致细胞生长失控。在这项提案的整个资助期内，将获得有关信息，以了解一般转录因子TFIIB参与基因激活的过程。为了了解激活蛋白是如何工作的，已经设计了一种遗传策略来识别Pho4和Adr1转录激活子的生理靶点。这两种蛋白都与TFIIB相互作用，作为其激活机制的一部分。该方法依赖于分离这些基因缺失导致的激活缺陷的基因外抑制物。为了分析TFIIB在转录中的一般作用，将进行全基因组微阵列分析。该实验将利用位于E62和R78位置的TFIIB突变体，这些突变体在转录起始位点选择中产生下游转移。为了阐明TFIIB E62和R78位突变如何影响基因表达的分子基础，将通过圆二色谱(CD)分析和分析它们对蛋白酶的敏感性来研究这些突变体的构象完整性。

DESCRIPTION (provided by applicant): Gene expression is regulated mainly at the level of transcription and transcription in turn, is regulated primarily at the level of initiation. Proper gene regulation is a critical event to maintain the homeostatic state of an organism. This is exemplified by diseases such as cancer, where improper gene regulation leads to uncontrolled cellular growth. Throughout the funding period of this proposal relevant information towards understanding the involvement of the general transcription factor TFIIB in the process of gene activation will be obtained. To comprehend how activator proteins work, a genetic strategy to identify physiological targets of the Pho4 and Adr1 transcription activators has been devised. Both proteins interact with TFIIB as part of their activation mechanism. The approach relies in the isolation of extragenic suppressors of the activation defect conferred by the deletion of these genes. To analyze the general role of TFIIB in transcription, a whole genome microarray analysis will be performed. The experiment will utilize TFIIB mutants at positions E62 and R78 that produce downstream shifts in transcription start site selection. To shed some light on the molecular basis of how do mutations at positions E62 and R78 of TFIIB affect gene expression, the conformational integrity of these mutants will be studied by means of circular dichroism (CD) analysis and by analyzing their susceptibility to proteases.