POLYSOMES & SUBUNITS: STRUCTURE-FUNCTION RELATIONSHIPS

本研究的目的是确定和 鉴定E.大肠杆菌核糖体RNA 在蛋白质合成的过程中。 使用质粒携带的rrnB 操纵子，我们正在16 S和23 S rRNA基因中产生突变， 体外和体内方法。 这些突变将由 各种分子和生化技术。 位点 感兴趣的包括3'16 S rRNA附近的Shine-Dalgarno区域 与mRNA碱基配对并可能参与移码， 16 S rRNA中识别翻译的潜在位点 mRNA中的增强子，16 S和23 S rRNA中参与亚基的区域 关联，以及16 S rRNA中可能涉及 在30 S亚基的活性和非活性形式之间切换。 待研究的其他区域包括假定的tRNA结合和 解码位点以及用于错义抑制的潜在位点， 16 S和23 S rRNA中赋予抗生素抗性的区域 例如壮观霉素、链霉素、氨基糖苷， 硫链丝菌素，最后是16 S rRNA中涉及的区域 翻译的终止。 致命的突变体将产生在 含有阻遏型启动子PL和T7的质粒。 质粒- 编码的rRNA将以大细胞以及 各种功能测定，其测量 体内翻译，包括无义和错义抑制。 方法将用于特异性分离质粒编码的 突变核糖体的结构和功能研究，使用沉默 16 S（1192位）和23 S（1067位）rRNA突变， 分别对壮观霉素和硫链丝菌素耐药。 这些 这些研究应该有助于我们理解 rRNA在蛋白质合成过程中的作用，通过定义催化 在分子水平上的RNA的特定区域的活性。

The objective of this research proposal is to identify and characterize functional sites in E. coli rRNA which are involved in the process of protein synthesis. Using a plasmid-borne rrnB operon we are producing mutations in the 16S and 23S rRNA genes by in vitro and in vivo methods. These mutations will be studied by a variety of molecular and biochemical techniques. Sites of interest include the Shine-Dalgarno region near the 3' 16S rRNA that base-pairs to mRNA and may bs involved in frameshifting, a potential site in 16S rRNA for the recognition of a translational enhancer in mRNA, regions in 16S and 23S rRNA involved in subunit association, and sites in 16S rRNA potentially involved in switching between the active and inactive forms of the 30S subunit. Additional regions to be studied include putative tRNA binding and decoding sites as well as potential sites for missense suppression, regions in 16S and 23S rRNA which confer resistance to antibiotics such as spectinomycin, streptomycin, the amino-glycosides, and thiostrepton, and finally regions in 16S rRNA which are involved in termination of translation. Lethal mutants will bs produced on plasmids containing repressible promoters PL and T7. The plasmid- coded rRNAs will be characterized by maxicells as well as by a variety of functional assays which measure rate and fidelity of translation in vivo, including nonsense and missense suppression. Methods will be used to specifically isolate the plasmid-coded mutant ribosomes for structural and functional studies using silent mutations in 16S (at 1192) and 23S (at 1067) rRNA that confer resistance to spectinomycin and thiostrepton, respectively. These studies should contribute to our understanding of the functional role of rRNA during protein synthesis by defining the catalytic activity of particular regions of the RNA at a molecular level.