POLYSOMES & SUBUNITES STRUCTURE-FUNCTION RELATIONSHIPS

我们将研究细菌核糖体的结构和功能。 利用16S和23S rRNA的突变体。重组人中的rrnB顺反子 将在选定的位置使用合成的方法诱变质粒 脱氧寡核苷酸与M13克隆技术。有待改进的领域 研究包括核糖体蛋白和信使核糖核酸结合部位，区域 亚基相互作用、肽基转移酶部位和结合部位 某些抗生素。突变的基因产物将在体内表征 一种改良的Maxicell体系及其体外分离 硫脲汞琼脂糖亲和层析、凝胶电泳法 蔗糖梯度离心法。这种遗传方法将提供 关于rRNA特定区域的特定信息，并增加我们的 了解rRNA在蛋白质合成中的重要作用。这个 核糖体蛋白S1在30S和70S核糖体上的结构排列 将使用针对S1和凝胶的单抗进行研究 免疫电泳法。这是一种方法的第一次应用 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-蛋白质-rrna相互作用的特征。初级结合蛋白 将被识别，rRNA上的特定连接位置将被 下定决心。这将为后续工作提供基本的结构信息 机能研究。EIF2在真核蛋白调控中的作用 合成将在酵母中进行研究。Alpha亚基被磷酸化 最近分离的酶eIF2-Kinase。这种酶将被鉴定为 并确定了调节其激活的因素。另一项监管规定 我们将探讨eif2-激酶的作用，即r-蛋白的磷酸化。 特别关注磷酸化对YL4，5S RNA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.