SITE-SPECIFIC MUTATION IN RRNA AND RIBOSOME FUNCTION

RRNA 和核糖体功能的位点特异性突变

• 批准号: 3438348
• 负责人: MELVIN SANTER
• 金额: $ 7.2万
• 依托单位: HAVERFORD COLLEGE
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1989-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3438348
• 关键词: Escherichia coli; bacterial genetics; bacteriophage M13; frameshift mutation; gel electrophoresis; genetic translation; mutant; nucleic acid sequence; ribosomal RNA; ribosomes

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.

核糖体是所有细胞中蛋白质合成的细胞器。 核糖体是一种大的核糖核蛋白复合物， 许多蛋白质，通常是三个大的RNA分子。 的 这些大分子的有组织的相互作用是 蛋白质合成（翻译）的反应，并决定 过程的准确性。 结构研究的目标是 核糖体是为了了解蛋白质和核糖核酸 酸在翻译 了解正常蛋白质合成 也许能帮助我们理解异常的生长过程 我们 特别是对rRNA在翻译中的作用感兴趣。 为 例如，有证据表明，16 S rRNA，一种分子1542 核苷酸长，在以下方面发挥着重要作用： 核糖体功能 看来，不同的职能作用， 16 S rRNA的结构由一级结构决定。 我们打算在各个地区创建特定于站点的基础更改 直接测试这些核苷酸在 蛋白质合成。 为此，我们使用了一些 序列仅相差一个碱基的脱氧寡核苷酸 16 S rRNA的野生型区域。 这些 寡核苷酸将被用作引物，单链寡核苷酸将被用作引物。 以噬菌体M13的DNA为模板，将其克隆到其中 16 S rDNA的特定区域。 体外合成突变体 DNA链将用于感染宿主细胞， 突变的rDNA分子。 突变体的相关区域 rDNA分子将被连接到各种质粒中， 生长在E.大肠杆菌，其中突变的功能测试 可以进行rRNA基因。 为了检测突变rRNA对翻译的影响， 准确地说，突变体rRNA将被放置在E.大肠杆菌携带 LacZ基因无义或移码突变， 抑制基因t-RNA。 翻译的增强或限制 将是衡量突变rRNA对准确性的影响 的翻译。 为了测试30 S核糖体的能力， 突变rRNA（1）参与30 S与50 S的结合 核糖体，和，（2）回收游离30 S核糖体，质粒 将在“maxicell”系统中使用含有突变rRNA的细胞。

项目成果

Base changes at position 792 of Escherichia coli 16S rRNA affect assembly of 70S ribosomes.

大肠杆菌 16S rRNA 792 位碱基变化会影响 70S 核糖体的组装。

• DOI: 10.1073/pnas.87.10.3700
• 发表时间: 1990
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Santer,M;Bennett-Guerrero,E;Byahatti,S;Czarnecki,S;O'Connell,D;Meyer,M;Khoury,J;Cheng,X;Schwartz,I;McLaughlin,J
• 通讯作者: McLaughlin,J