GENETIC ANALYSIS OF BACTERIOPHAGE T4 REGA PROTEIN-RNA

噬菌体T4的RegA蛋白是一种翻译抑制物。 几个T4信使核糖核酸。遗传方法在本文件中描述 将识别RegA蛋白的氨基酸的提案 接触mR NA结合位点。在原则项目中， 允许受控制的RegA表达的质粒将是 在体外和体内诱变，然后将筛选出 含RegA敏感菌株中RegA功能的改变 基因或敏感基因融合。动力学描述将是 为减少的、紧密结合的或新的RegA蛋白获得的 特定部位的表型，然后将与单个 氨基酸替代(通过核酸测序确定) 每个抑制子突变体。氨基酸残基和短链 RegA已被选为靶向突变。从 使用随机突变的初步结果和所描述的新的 选择技术，特别感兴趣的RegA残留物将是 被确定用于进一步的定向突变研究。 Rega函数的两个方面被视为以下较小的工作 这项提议。一个翻译激活函数已经被 暂时归因于RegA蛋白；激活的蛋白 将通过双向凝胶电泳法进行鉴定和测序。这个 RegA mRNA结合部位也会呈现随机性 与An合作的寡核苷酸合成 建立了致力于翻译调控的实验室。这些 这些努力将补充我们上述的主要利益。 实验室的长期目标是确定rna-蛋白质。 RegA翻译抑制因子与细胞外信号转导通路的相互作用 预期Rega的功能将与 了解其他原核生物和动物的RNA-蛋白质相互作用 真核系统。

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.