ASSEMBLING AN UNDERSTANDING OF RIBOSOME BIOSYNTHESIS

描述(改编自申请者摘要)：约40% 大肠杆菌产生的总能量被消耗在合成大型 核糖体成分的数量，3个总超过4000个核苷酸的RNA和 超过50种蛋白质。在细菌中，细胞生长和核糖体的速率 生物合成是密切相关和直接相关的。因此，对于单元格 核糖体成分的合成及其随后的组装 必须协调一致。这些观察表明，一个准确组装的 而功能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。尽管，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(S)和动力学在很大程度上仍未确定 尽管对细胞生理学具有普遍的重要性。 这一建议描述了三种研究机制、途径和 核糖体组装的动力学。这项建议的目标是了解 影响大肠杆菌30S核糖体亚基的因素 集合。第一，使用一套完整的30S亚基的体外重建 重组小亚基核糖体蛋白将用于鉴定16S 参与30S亚基的rRNA核苷酸和小亚基核糖体蛋白 集合。第二，溶液化学探测和小亚基的结合 将使用核糖体蛋白定向的羟基自由基探针来定位16S 30S亚基形成不同阶段的rRNA折叠。最后，在体外 将使用重组和标准生化方法来净化和 描述参与核糖体组装的细胞(核糖体外)因子。 初步结果表明，这三个具体目标都将增加我们的 了解核糖体组装。从这些研究中获得的见解将使 美国将继续进行未来研究体内核糖体生物发生的实验。在……里面 此外，这些研究可能确定调节细菌细胞的靶标。 这可能会导致新型抗菌剂的生产。

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.