Assemblying an Understanding of Ribosome Biosynthesis

描述(申请人提供)：大约40%的大肠杆菌总能量生产被消耗来合成大量的核糖体成分：3个RNA(由400多个核苷酸组成)和50多个蛋白质。这一观察结果以及大肠杆菌生长速度和核糖体生物发生之间的直接相关性表明，准确组装和功能的核糖体对细胞活力至关重要。我们的长期目标是详细了解核糖体的生物发生，从而揭示这一动态过程对细胞生理的基本方面的影响，如生长调节。本提案详细介绍了对大肠杆菌小核糖体亚单位生物发生的研究。这项工作的目的是深入了解SOS功能亚基组装过程中的构象变化和调节这些变化的因素。已经提出了三个具体目标来解决这些问题。首先，16S rRNA和核糖体蛋白在组装的不同阶段促进这些变化的构象变化将使用一组化学探测策略来识别。其次，利用遗传学和生物化学相结合的方法，将鉴定和表征体内形成的新的SOS亚单位组装中间体。两个核糖体蛋白突变株，包括核糖体蛋白S15的染色体缺失，正在进行这项分析。第三，分析促进SOS亚基组装的核糖体外因子。将详细探讨在当前供资期间确定的因素，新方法将导致确定和确定其他因素的特征。我们在当前资金周期中的进展和我们的初步数据，包括生化和遗传数据，表明所有三个特定目标都将成功，因此将直接影响我们对SOS亚单位生物发生的理解。这样的理解对于确定抑制细菌生长的新靶点至关重要，因此对于开发抗菌剂也是至关重要的。

DESCRIPTION (provided by applicant): Approximately 40% of the total energy production of E. coli is consumed to synthesize the large number of ribosomal components: 3 RNAs (comprised of more than 400 nucleotides) and greater than 50 proteins. This observation and the direct correlation between the rates of E. coli growth and ribosome biogenesis indicate that an accurately assembled and functional ribosome is of utmost importance for cell viability. Our long term goal is to gain a detailed understanding of ribosome biogenesis and thus reveal the impact of this dynamic process on fundamental aspects of cell physiology, such as growth regulation. This proposal details studies of E. coli small (SOS) ribosomal subunit biogenesis. The purpose of this work is to gain an in-depth understanding of conformational changes and the factors that modulate these changes during the course of functional SOS subunit assembly. Three specific aims have been proposed to address these questions. First, conformational changes in 16S rRNA and the ribosomal proteins that facilitate these changes during different stages of assembly will be identified using a panel of chemical probing strategies. Second, using a combination of genetics and biochemistry, novel SOS subunit assembly intermediates formed in vivo will be identified and characterized. Two ribosomal protein mutant strains, including a chromosomal deletion of ribosomal protein S15, are in hand for this analysis. Third, extra- ribosomal factors that facilitate SOS subunit assembly will be analyzed. Factors identified in the current funding period will be explored in detail and new methods will lead to the identification and characterization of additional factors. Our progress during the current funding cycle and our preliminary data, both biochemical and genetic, indicate that all three specific aims will be successful and thus will directly impact our understanding of SOS subunit biogenesis. Such an understanding is critical for the identification of novel targets for attenuating bacterial growth and therefore for the development of antimicrobials.