ASSEMBLING AN UNDERSTANDING OF RIBOSOME BIOSYNTHESIS

加深对核糖体生物合成的理解

• 批准号: 6231344
• 负责人: Gloria M Culver
• 金额: $ 20.71万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6231344
• 关键词: Escherichia coli; SDS polyacrylamide gel electrophoresis; bacterial RNA; bacterial proteins; biosynthesis; chemical aggregate; intermolecular interaction; microorganism culture; nucleic acid reconstitution; nucleic acid structure; protein folding; protein reconstitution; recombinant proteins; ribosomal RNA; ribosomal proteins; ribosomes

DESCRIPTION (adapted from applicant's abstract): Approximately 40 percent of the total energy production of E. coli is consumed to synthesize the large number of ribosomal components, 3 RNAs totaling over 4000 nucleotides and greater than 50 proteins. In bacteria, the rates of cell growth and ribosome biosynthesis are intimately related and directly correlated. Thus, for cell viability, the synthesis of ribosomal components and their subsequent assembly must be coordinated. These observations suggest that an accurately assembled and functional ribosome is one of the most important cellular organelles and that defects in ribosome assembly would be fatal. Although, years of research have been dedicated to studying ribosomal components and ribosome function, the pathway(s) and dynamics of ribosome assembly remain largely uncharacterized despite general importance to cell physiology. This proposal describes three approaches to study the mechanism, pathway and dynamics of ribosome assembly. The goal of this proposal is to understand what factors are involved in and critical for E. coli 30S ribosomal subunit assembly. First, in vitro reconstitution of 30S subunits using a complete set of recombinant small subunit ribosomal proteins will be used to identify l6S rRNA nucleotides and small subunit ribosomal proteins involved in 30S subunit assembly. Second, a combination of solution chemical probing and small subunit ribosomal protein-directed hydroxyl radical probing will be used to map 16S rRNA folding during different stages of 30S subunit formation. Lastly, in vitro reconstitution and standard biochemical approaches will be used to purify and characterize cellular (extra-ribosomal) factors involved in ribosome assembly. Preliminary results suggest that all three specific aims will add to our understanding of ribosome assembly. Insights gained in these studies will allow us to pursue future experiments studying ribosome biogenesis in vivo. In addition, these studies may identify targets for regulation of bacterial cell growth and perhaps lead to the production of novel antimicrobials.

描述（改编自申请人的摘要）：大约 40% 大肠杆菌产生的总能量被消耗来合成大量的 核糖体成分的数量，3个RNA，总计超过4000个核苷酸， 超过50种蛋白质。在细菌中，细胞生长和核糖体的速率 生物合成密切相关且直接相关。因此，对于细胞 活力、核糖体成分的合成及其随后的组装 必须协调一致。这些观察结果表明，精确组装的 功能性核糖体是最重要的细胞器之一 核糖体组装的缺陷将是致命的。虽然经过多年的研究 一直致力于研究核糖体成分和核糖体功能， 核糖体组装的途径和动力学在很大程度上仍然未知 尽管对细胞生理学具有普遍重要性。 该提案描述了研究机制、途径和方法的三种方法 核糖体组装动力学。该提案的目标是了解什么 参与大肠杆菌 30S 核糖体亚基且对其至关重要的因素 集会。一、30S亚基使用全套体外重建 重组小亚基核糖体蛋白将用于鉴定 l6S 参与 30S 亚基的 rRNA 核苷酸和小亚基核糖体蛋白 集会。二、溶液化学探测与小亚基的结合 核糖体蛋白定向羟基自由基探测将用于绘制 16S 图谱 rRNA 在 30S 亚基形成的不同阶段折叠。最后，在体外 将使用重构和标准生化方法来纯化和 表征参与核糖体组装的细胞（核糖体外）因子。 初步结果表明，所有三个具体目标都将增加我们的 了解核糖体组装。在这些研究中获得的见解将允许 我们将继续进行未来研究体内核糖体生物发生的实验。在 此外，这些研究可能会确定细菌细胞调节的靶标 增长并可能导致新型抗菌剂的生产。