POLYSOMES & SUBUNITS: STRUCTURE-FUNCTION RELATIONSHIPS

这项研究建议的目标是确定和 鉴定E.ColirRNA中涉及的功能位点 在蛋白质合成的过程中。使用质粒携带的rrnB 操纵子我们正在产生16S和23S rRNA基因的突变 体外和体内实验方法。这些突变将由以下人员研究 各种分子和生化技术。网站： 感兴趣的包括3‘16S rRNA附近的Shine-Dalgarno区域 该碱基对连接到信使核糖核酸，并可能参与框架移位，a 16S rRNA中识别翻译基因的潜在位点 亚基涉及的16S和23S rRNA区域的mRNA增强子 关联，以及16S rRNA中可能参与的位点 在30s亚基的活性形式和非活性形式之间切换。 其他有待研究的区域包括假定的tRNA结合和 解码位置以及用于错义抑制的潜在位置， 16S和23S rRNA中对抗生素产生耐药性的区域 如壮观霉素、链霉素、氨基糖苷和 硫链霉菌，最后是16S rRNA中涉及的区域 在翻译终止时。致命的突变体将在 含有阻遏启动子PL和T7的质粒。该质粒- 编码的rRNA的特征是最大细胞以及一个 各种功能分析，衡量的速度和保真度 体内翻译，包括无义和错义抑制。 方法将被用来专门分离编码的质粒 突变核糖体在结构和功能研究中的应用 16S(1192)和23S(1067)rRNA突变 对壮观霉素和硫链霉菌分别耐药。这些 研究应该有助于我们理解功能 RRNA在蛋白质合成中的作用 核糖核酸特定区域在分子水平上的活性。

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.