FUNCTIONAL ANALYSIS OF TFIIB PROTEIN IN S CEREVISIAE

描述：该项目的长期目标是了解 真核生物基因激活的分子机制研究 酿酒酵母中的转录因子TFIIB。在……里面 这个实验室之前的工作，物种特定区域的突变 TFIIB的表达被证明影响了一个报告基因的表达 激活剂HAP4，但不是非激活的转录或激活 GAL4.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。 在这项申请中提出了三个具体目标。具体目标1将 检验TFIIB基因物种特异性区域突变的假设 影响HAP4与TFIIB之间的相互作用，从而削弱HAP4与TFIIB的作用能力 HAP4激活转录。生化和遗传学研究都将 来直接检验这一假设。《特定目标2》将测试 假设TFIIB的其他区域参与了 体内的基因激活。TFIIB突变体将被寻找，无论是哪一种 响应激活器或绕过激活器要求。它是 希望这些突变体的特征将有助于揭示更多 体内基因激活的关键步骤或事件。具体目标3将 检验这样一种假设，即额外的蛋白质对转录和 温度抑制因子的筛选可以揭示基因的激活 敏感的yTFIIB突变体。这类病毒的分离和鉴定 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.