MECHANISMS OF ACTION OF YEAST TRANSCRIPTIONAL REGULATORS

这项工作的目标是了解，在精确的分子术语， 酵母转录抑制因子α 2的作用机制 蛋白 这种蛋白质关闭了一组酵母的转录 细胞类型特异性基因称为a-特异性基因。 阿尔法2蛋白质紧密而特异地结合在位于 在α-特异性基因STE 6的上游。 这个操作员带来了 当位于“测试”启动子内或完全位于“测试”启动子的上游时，抑制。 为解释这种远距离抑制而开发的具体模型将在 通过所提出的实验进行严格测试。 具体地，可以确定启动子的距离（相对于测试启动子）。 操作员可以确定功能。 限制（如有） 将定义阻遏所需的启动子/操纵子几何结构。 突变体 将生成运算符以确定运算符是否只是alpha 2识别序列，或如所怀疑的，包含一些额外的特征 需要镇压。 将毫克量的α 2纯化至均匀，并用于 确定Alpha 2是否在绑定到其操作符时展开 DNA和/或弯曲DNA。 分子探针实验将揭示 Alpha 2-operator相互作用的结构特征。 纯化的α 2蛋白将用作亲和试剂， 尝试识别、纯化和表征涉及的其他组分 镇压机制。 这些其他成分的生理作用 都是从基因实验中推导出来的 阿尔法2与第二个调节因子结合作用的机制 蛋白质A1将被测定。 这些蛋白质一起关闭 一组被称为单倍体特异性基因的基因的转录。 我们 将纯化α 2/α 1调节活性，确定其组成 并研究其性质。 最后，酵母转录系统将在体外重建， 为了确定转录中的一个或多个精确步骤， 阿尔法2号的行动 对阿尔法2的拟议研究将提供详细的分子图像 显示了酵母中细胞的特化是如何维持的 啤酒。 基本了解细胞内的分子事件 专业化对于理解流程如何失败至关重要 引起病理状况。

The goal of this work is to understand, in precise molecular terms, the mechanism of action of a yeast transcriptional repreessor, the Alpha 2 protein. This protein turns off transcription of a group of yeast cell-type specific genes called the a-specific genes. The Alpha 2 protein binds tightly and specifically to an operator located upstream of an a-specific gene, STE6. This operator brings about repression when located within or entirely upstream of a "test" promoter. Specific models developed to explain this repression at a distance will be rigorously tested by the proposed experiments. Specifically, the distance (relative to the test promoter) over which the operator can function will be determined. Constraints (if any) on the promoter/operator geometry required for repression will be defined. Mutant operators will be generated to determine if the operator is simply an alpha 2 recognition sequence or, as suspected, contains some additional feature required for repression. Milligram quantities of Alpha 2 will be purified to homogeneity and used to determine whether or not Alpha 2, when it binds to its operator, unwinds the DNA and/or bends the DNA. Molecular probe experiments will reveal structural feature of the Alpha 2-operator interaction. The purified Alpha 2 protein will be used as an affinity reagent in an attempt to identify, purify and characterize other components involved in the repression mechanism. The physiological role of these other components will be deduced from genetic experiments. The mechanism by which Alpha 2 acts in combination with a second regulatory protein, a1, will be determined. Together, these proteins turn off transcription of a group of genes known as the haploid-specific genes. We will purify the Alpha 2/a1 regulatory activity, determine its composition and study its properties. Finally, a yeast transcription system will be reconstituted in vitro in order to determine the precise step or steps in transcription blocked by the action of Alpha 2. The proposed studies of Alpha 2 will provide a detailed molecular picture showing how cell specialization is maintained in the yeast Saccharomyces cerevisiae. A basic understanding of the molecular events underlying cell specialization is essential for understanding how the process can fail giving rise to pathological conditions.