GENETIC ANALYSIS OF A TRANSLATIONAL REPRESSOR

蛋白质-RNA相互作用日益被认识到对 各种基本的细胞活动。RNA的外壳蛋白 噬菌体MS2是一种翻译抑制因子。在……末期 感染性外壳蛋白与病毒中特定的茎环结构结合 复制酶的信使核糖核酸和应答合成。尽管我们已经知道很多 21个核苷酸运算符的重要功能成分 序列，关于外壳蛋白的类似信息是不可用的。我们有 构建了一种遗传系统，在该系统中外壳蛋白抑制了 复制酶-β-半乳糖苷酶融合蛋白。这使我们能够 方便地应用分子遗传学的方法来确定 以外壳蛋白为靶标的蛋白质-RNA特异性相互作用的分子基础 一个模范系统。具体地说，我们希望实现以下目标 目标： 1.确定外壳蛋白与RNA结合所需的氨基酸残基 通过分离和鉴定一系列改变 操纵子结合的强度和/或特异性。我们将隔离 以下类型的突变： A.抑制子缺陷突变， B.抑制子缺陷突变的基因内抑制物， 和 C.操纵子结构的外壳蛋白抑制因子 突变。 2.突变的外壳蛋白的RNA结合特性将是 其特点是： A.在体内通过测量抑制合成的 复合复制酶-β-半乳糖苷酶， B.体外测定RNA结合亲和力，以及 C.通过化学修饰研究，将确定 运算符中的核苷酸对 野生型和突变型外壳蛋白的识别。 3.从遗传学和生化学的角度检验这样一种观点，即 外壳蛋白巯基与必需氨基酸之间的瞬时共价键 操纵子中的嘧啶是外壳蛋白-RNA的一个组成部分 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.