FUNCTIONAL ANALYSIS OF TFIIB PROTEIN IN S CEREVISIAE

描述：本项目的长期目标是了解 真核生物基因激活的分子机制， 转录因子TFIIB在酵母，酿酒酵母。 在 这个实验室以前的工作，在一个物种特异性区域的突变， TFIIB的表达被证明会影响由TFIIB激活的报告基因的表达。 激活因子HAP4，但不激活转录或激活 GAL 4. 这为yTFIIB参与的想法提供了强有力的支持 活细胞中基因激活的过程。 在本申请中提出了三个具体目标。 具体目标1将 检验TFIIB物种特异性区域突变的假设 影响HAP 4和TFIIB之间的相互作用，从而削弱了HAP 4和TFIIB之间的相互作用。 HAP4激活转录。 生物化学和遗传学研究将 直接测试这个假设。 具体目标2将测试 假设TFIIB的其他区域参与了 体内基因激活。 将寻找TFIIB突变体，其不能 响应激活器或绕过对激活器的要求。 是 希望这些突变体的特征将有助于揭示更多的 体内基因激活的关键步骤或事件。 第3章将 测试的假设，额外的蛋白质重要的转录和 基因激活可以通过筛选温度抑制因子来揭示 敏感的yTFIIB突变体。 分离和表征这种 转录因子将进一步决定TFIIB突变体如何影响 体内基因激活的过程。

DESCRIPTION: The long term goal of this project is to understand the molecular mechanisms of gene activation in eukaryotes using the general transcription factor TFIIB in the yeast, Saccharomyces cerevisiae. In previous work from this laboratory, mutations in a species-specific region of TFIIB were shown to affect the expression of a reporter gene activated by the activator HAP4, but not non-activated transcription or activation by GAL4. This provides strong support for the idea that yTFIIB participates in the process of gene activation in living cells. Three specific aims are proposed in this application. Specific Aim 1 will test the hypothesis that mutations in the species-specific region of TFIIB affect the interaction between HAP4 and TFIIB, thus weakening the ability of HAP4 to activate transcription. Both biochemical and genetic studies will be conducted to test this hypothesis directly. Specific Aim 2 will test the hypothesis that additional regions of TFIIB participate in the process of gene activation in vivo. TFIIB mutants will be sought that either fail to respond to activators or that bypass the requirement for activators. It is hoped that the characterization of these mutants will help reveal additional steps or events critical for gene activation in vivo. Specific Aim 3 will test the hypothesis that additional proteins important for transcription and gene activation can be revealed by screens for suppressors of temperature sensitive yTFIIB mutants. The isolation and characterization of such transcription factors will further determine how the TFIIB mutants affect the process of gene activation in vivo.