GENETIC ANALYSIS OF BACTERIOPHAGE T4 REGA PROTEIN-RNA

噬菌体T4 regA蛋白是一种翻译阻遏物， 几个T4 mRNA。 遗传学方法在此描述 该提案将确定regA蛋白的氨基酸， 接触mRNA结合位点。 在原则项目中， 允许受控regA表达的质粒将被 在体外和体内诱变，然后筛选 在E.含有regA敏感的大肠杆菌菌株 基因或敏感基因融合。 动力学描述将是 获得regA蛋白减少，紧密结合或新的 位点特异性表型，然后将其与单个 氨基酸取代（通过核酸测序确定）， 每个阻遏突变体。 氨基酸残基和短区域 regA已被选择用于靶向诱变。 从 使用随机诱变的初步结果和所描述的新的 选择技术，特别感兴趣的regA残基将被 鉴定用于进一步定向诱变研究。 regA函数的两个方面作为一个较小的努力来解决， 这个提议。 翻译激活功能已经被 暂时归因于regA蛋白;活化的蛋白质 将通过2D凝胶电泳鉴定并测序。 的 regA mRNA结合位点的特征还在于随机 寡核苷酸合成的合作努力， 建立了翻译调控实验室。 这些 这些努力将补充我们上述的主要利益。 该实验室的长期目标是定义RNA蛋白 regA翻译阻遏物与 期望regA的功能与以下内容相关： 了解其他原核生物的RNA蛋白质相互作用， 真核生物系统

The bacteriophage T4 regA protein is a translational repressor of several T4 mRNAs. Genetic methods are described in this proposal that will identify amino acids of the regA protein that contact the mRNA binding sites. In the principle project, plasmids that allow controlled regA expression will be mutagenized in vitro and in vivo and will then be screened for altered regA function in E. coli strains containing regA-sensitive genes or sensitive gene fusions. Kinetic descriptions will be obtained for regA proteins with diminished, tight-binding or new site-specifity phenotypes that will then be correlated to the single amino acid substitution (determined by nucleic acid sequencing) in each repressor mutant. Amino acid residues and short regions of regA have been selected for targeted mutagenesis. From the initial results using random mutagenesis and the described new selection techniques, regA residues of particular interest will be identified for further directed mutagenesis studies. Two aspects of regA function are addressed as a smaller effort of this proposal. A translational activation function has been tentatively ascribed to the regA protein; the activated proteins will be identified by 2D gel electrophoresis and sequenced. The regA mRNA binding site will also be characterized by random oligonucleotide synthesis in a collaborative effort with an established laboratory working on translational regulation. These efforts will complement our principle interests described above. The long-term goal of the laboratory is to define the RNA-protein interactions of the regA translational repressor with the expectation that features of regA will be relevant to understanding RNA-protein interactions of other prokaryotic and eukaryotic systems.