SITE-SPECIFIC MUTATION IN RRNA AND RIBOSOME FUNCTION

核糖体是所有细胞中合成蛋白质的细胞器。 核糖体是一种大的核糖核蛋白复合体，由 许多蛋白质，通常还有三个大的RNA分子。这个 这些大分子的有组织的相互作用是 蛋白质合成的反应(翻译)，并决定 过程的准确性。结构研究的目标是 核糖体是理解蛋白质和核糖核的作用 翻译中的酸。对正常蛋白质合成的理解 可能会帮助我们理解异常的生长过程。我们是 对rRNA在翻译中的作用特别感兴趣。为 例如，有证据表明，16S rRNA，一个分子1542 核苷酸很长，在许多方面发挥着重要作用 核糖体功能。似乎不同的功能作用不同 16S rRNA的区域由一级结构决定。 我们打算在不同地区创建特定于站点的基础更改 直接测试这些核苷酸在细胞周期中的作用 蛋白质合成。为此，我们使用了许多 序列上只有一个碱基不同的脱氧寡核苷酸 来自16S rRNA的野生型区域。这些 寡核苷酸将作为引物，与单链 以噬菌体M13为模板，克隆入噬菌体M13 16S rDNA的特定区域。体外合成的突变体 DNA链将被用来感染宿主细胞，这将产生 突变的rDNA分子。该突变体的相关区域 RDNA分子将连接到各种质粒中，这些质粒可以 在大肠杆菌中生长，在那里测试突变的功能 RRNA基因是可以携带的。 为了测试突变rRNA对翻译的影响 准确地说，突变的rRNA将被置于携带有 LacZ基因的无义或移码突变和 抑制子t-RNA。加强或限制翻译 将是衡量突变rrna对精确度的影响的指标。 翻译的问题。测试含有30S核糖体的能力 突变rRNA将(1)参与30s与50s的关联 核糖体，以及，(2)作为游离的30S核糖体，质粒循环 含有突变的rRNA将被用于“Maxicell”系统。

Ribosomes are the organelles of protein synthesis in all cells. Ribosomes are large ribonucleoprotein complexes composed of many proteins and, usually, three large RNA molecules. The organized interaction of these macromolecules underlies the reactions of protein synthesis (translation) and determines the accuracy of the process. The goal of structural studies on ribosomes is to understand the role of proteins and ribonucleic acid in translation. An understanding of normal protein synthesis may help us to understand aberrant growth processes. We are particularly interested in the role of rRNA in translation. For example, there is evidence that 16S rRNA, a molecule 1542 nucleotides long, performs a number of important roles in ribosome function. It appears that the functional role of different regions of 16S rRNA is determined by the primary structure. We intend to create site-specific base changes in various regions of 16S rRNA to directly test the role of those nucleotides in protein synthesis. To do this, we use a number of deoxyoligonuleotides which differ in sequence by only one base from the wild-type region of 16S rRNA in question. These oligonucleotides will be used as a primer, with the single-strand DNA of the phage M13 as template, which has cloned into it specific regions of 16S rDNA. The in vitro synthesized mutant DNA strand will be used to infect host cells which will generate mutant rDNA molecules. The relevant regions of the mutant rDNA molecules will be ligated into various plasmids which can be grown in E. coli, where the test for the function of the mutated rRNA gene can be carried out. To test for the effect of a mutant rRNA on translational accuracy, the mutant rRNA will be placed in an E. coli carrying a nonsense or frameshift mutation in the LacZ gene and a suppressor t-RNA. Enhancement or restriction of the translation will be a measure of the effect of the mutant rRNA on accuracy of translation. To test for the ability of 30S ribosomes containing mutant rRNA to (1) participate in 30S association with 50S ribosomes, and, (2) to recycle as free 30S ribosomes, plasmids containing mutant rRNA will be used in the "maxicell" system.