MECHANISMS OF ACTION OF YEAST TRANSCRIPTIONAL REGULATORS

这项工作的目标是用精确的分子术语来理解 酵母转录复制子Alpha2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被称为a-特异性基因。 Alpha 2蛋白紧密结合并特异性地与位于 位于a特异性基因STE6的上游。这个运算符带来了 位于“测试”启动子或完全位于启动子上游的抑制基因。 为解释这种远距离压制而开发的特定模型将是 通过拟议的实验进行了严格的测试。 具体地说，距离(相对于测试启动子) 操作员能否发挥作用将被确定。上的约束(如果有) 将定义抑制所需的启动器/操作员几何形状。突变型 将生成运算符以确定该运算符是否只是一个字母 2识别序列，或如怀疑的，包含一些额外的特征 是镇压所必需的。 毫克量的阿尔法2将被提纯到均质，并用于 确定当Alpha 2与其操作符绑定时是否解开 DNA和/或使DNA弯曲。分子探针实验将揭示 阿尔法2-算符相互作用的结构特征。 纯化的Alpha2蛋白将作为亲和试剂用于 尝试识别、提纯和表征 压抑机制。这些其他成分的生理作用 将从基因实验中推断出来。 阿尔法2与第二调节因子结合的作用机制 蛋白质A1将被测定。在一起，这些蛋白质会关闭 转录一组被称为单倍体特异基因的基因。我们 将提纯Alpha2/A1调节活性，确定其组成 并研究它的性质。 最后，一个酵母转录系统将在体外重组 以确定转录中被阻止的一个或多个精确步骤 阿尔法2的行动。 对阿尔法2的拟议研究将提供详细的分子图景 酵母细胞特化是如何维持的 酿酒。对细胞内的分子事件有基本的了解 专业化对于理解流程失败的原因至关重要 引起病理状态的。

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.