GENETIC ANALYSIS OF A TRANSLATIONAL REPRESSOR

蛋白质-RNA相互作用越来越被认为是重要的， 各种重要的细胞活动。 RNA的外壳蛋白 噬菌体MS 2是一种翻译阻遏物。 晚些时候， 感染外壳蛋白结合病毒中的特定茎环结构， 复制酶的mRNA和反应合成。 虽然我们已经知道很多 21个核苷酸操纵子的重要功能成分 序列，关于外壳蛋白的类似信息是不可用的。 我们有 构建了一个遗传系统，其中外壳蛋白抑制合成的 复制酶-β-半乳糖苷酶融合蛋白。 这使我们能够 可方便地应用分子遗传学的方法来确定 利用外壳蛋白作为特异性蛋白质-RNA相互作用的分子基础 一个模型系统 具体而言，我们希望实现以下目标 目标： 1. 定义外壳蛋白RNA结合所需的氨基酸残基 通过分离和表征一些突变来发挥作用， 操作符绑定的强度和/或特异性。 我们将隔离 以下类型的突变： a. 阻遏物缺陷突变， B. 阻遏物缺陷突变的基因内阻遏物， 和 C. 操纵子组成型外壳蛋白抑制子 突变。 2. 突变体外壳蛋白的RNA结合特性将是 其特征在于： a. 在体内通过测量抑制的合成 杂合复制酶-β-半乳糖苷酶， B. RNA结合亲和力的体外测定，和 C. 通过化学改性研究， 操纵子中的核苷酸， 被野生型和突变体外壳蛋白识别。 3. 从遗传学和生物化学的角度来验证一种观点， 外壳蛋白巯基和必需的 嘧啶是外壳蛋白-RNA的组成部分 互动

Protein-RNA interactions are increasingly recognized as important to a variety of essential cellular activities. The coat protein of the RNA bacteriophage MS2 is a translational repressor. At late times of infections coat protein binds a specific stem-loop structure in the viral mRNA and responses synthesis of replicase. Although we already know many of the important functional components of the 21 nucleotide operator sequence, similar information about coat protein is unavailable. We have constructed a genetic system in which coat protein represses synthesis of a replicase-beta-galactosidase fusion protein. This permits us to conveniently apply the methods of molecular genetics to determine the molecular basis of specific protein-RNA interactions using coat protein as a model system. Specifically we want to accomplish the following objectives: 1. Define amino acid residues necessary for coat protein's RNA binding function by isolating and characterizing a number of mutations that alter the strength and/or specificity of operator binding. We will isolate mutations of the following type: a. repressor-defective mutations, b. intragenic suppressors of repressor-defective mutations, and c. coat protein suppressors of operator-constitutive mutations. 2. The RNA binding properties of mutant coat proteins will be characterized: a. in vivo by measuring repression of synthesis of the hybrid replicase-beta-galactosidase enzyme, b. in vitro determination of RNA binding affinity, and c. by chemical modification studies which will determine nucleotides in the operator that are important for recognition by wild-type and mutant coat proteins. 3. Test genetically and biochemically the idea that the formation of a transient covalent bond between a coat protein sulfhydryl and an essential pyrimidine in the operator is a component of the coat protein-RNA interaction.