POLYSOMES & SUBUNITES STRUCTURE-FUNCTION RELATIONSHIPS

研究细菌核糖体的结构和功能 使用16 S和23 S rRNA的突变体。 重组体中的rrnB顺反子 质粒将在选定的位点使用合成的 脱氧寡核苷酸和M13克隆技术。 领域得到 研究包括核糖体蛋白和mRNA结合位点， 亚基相互作用，肽基转移酶位点和结合位点， 某些抗生素 突变基因产物将在体内进行表征， 改良的Maxicell系统，并在体外分离后， 硫尿苷汞琼脂糖亲和层析，凝胶电泳 和蔗糖梯度离心。 这种遗传学方法将提供 关于rRNA特定区域的特定信息， 了解rRNA在蛋白质合成中的重要作用。 的 核糖体蛋白S1在30 S和70 S核糖体上结构排列 将使用S1的单克隆抗体和凝胶进行研究 免疫电泳 这是第一次应用一种方法， 用于表征许多核糖体蛋白质的精细结构。 酵母核糖体的结构-功能研究将集中在 R-蛋白-rRNA相互作用的表征。 初级结合蛋白 将被识别，rRNA上的特异性附着位点将被 测定 这将为后续研究提供基本的结构信息。 功能研究 eIF 2在真核蛋白调节中的作用 将在酵母中研究合成。 α亚基被磷酸化， 最近分离的eIF 2激酶。 酶将被表征为 并确定调节其活化的因素。 额外的监管 eIF 2-激酶的作用，r-蛋白的磷酸化，将被探讨 特别关注磷酸化对YL 4的影响， 结合蛋白 这些实验将增加我们对 共价修饰在真核生物翻译调控中的作用 细胞

The structure and function of the bacterial ribosome will be investigated using mutants in 16S and 23S rRNA. The rrnB cistron in a recombinant plasmid will be mutagenized at selected sites using synthetic deoxy-oligonucleotides and the M13 cloning technology. Areas to be investigated include ribosomal protein and mRNA binding sites, regions of subunit interaction, the peptidyl transferase site and binding sites for certain antibiotics. Mutant gene products will be characterized in vivo in a modified maxicell system and in vitro after isolation by thiouridine-mercurated agarose affinity chromatography, gel electrophoresis and sucrose gradient centrifugation. This genetic approach will provide specific information about particular regions of rRNA and increase our understanding of the significant role of rRNA in protein synthesis. The structural arrangement of ribosomal protein S1 on 30S and 70S ribosomes will be studied using monoclonal antibodies to S1 and gel immunoelectrophoresis. This is the first application of an approach to be used to characterize the fine structure of many ribosomal proteins. Structure-function studies of yeast ribosomes will focus on characterization of r-protein-rRNA interactions. Primary binding proteins will be identified and specific attachment sites on rRNA will be determined. This will provide basic structural information for subsequent function studies. The role of eIF2 in the regulation of eucaryotic protein synthesis will be studied in yeast. The Alpha subunit is phosphorylated by the recently isolated enzyme eIF2-kinase. The enzyme will be characterized and factors regulating its activation determined. An additional regulatory role of eIF2-kinase, the phosphorylation of r-proteins, will be explored with particular focus on the effect of phosphorylation on YL4, the 5S RNA binding protein. These experiments will increase our understanding of the role of covalent modification in regulation of translation in eucaryotic cells.