Assembly of E. coli ribosomes in vitro and in cells

大肠杆菌核糖体的体外和细胞内组装

• 批准号: 9205511
• 负责人: James R Williamson
• 金额: $ 47.35万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-02-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9205511
• 关键词: Anti-Infective Agents; Antibiotics; Bacteria; Binding; Biogenesis; Biological Assay; Cells; Escherichia coli; Event; Fluorescence; In Vitro; Isotopes; Kinetics; Label; Lead; Mass Spectrum Analysis; Measurement; Microfluidic Microchips; Microscope; Monitor; Physiologic pulse; Process; Protein Biosynthesis; Ribosomal Proteins; Ribosomes; Role; Son of Sevenless Proteins; Spectrum Analysis; Time; biophysical techniques; cofactor; experimental study; in vivo; insight; novel; stable isotope; two-photon

This proposal outlines a plan to investigate the mechanism of assembly of E. coli ribosomes both in vitro and in vivo. The ribosome is the macromolecular machine responsible fbr all protein synthesis in cells, and it is efficiently assembled from over 50 components. Using a combination of biophysical methods, we will Investigate the order of events in assembly and the kinetics of assembly in vitro. We will examine the role of assembly cofactors in E. coli, and examine the effects of antibiotics on the assembly process. We have developed a novel isotope pulse-chase assay that enables measurement of binding kinetics for ribosomal proteins using mass spectrometry. We are developing a two-photon fluorescence correlation microscope to monitor the assembly of fluorescently labeled ribosomal proteins in real time. Finally, we are extending our mass spectrometry analysis to isotope pulse experiments in living bacteria, which allows us to directly monitor the biogenesis of ribosomes. Taken together, these studies will provide new mechanistic insights into the critical process of ribosome assembly.

该建议概述了研究大肠杆菌核糖体组装机制的计划 和体内。核糖体是大分子机器负责FBR的FBR所有蛋白质合成，并且 它是从50多个组件中有效组装的。结合生物物理方法，我们将 研究体外组装和组装动力学中事件的顺序。我们将研究 大肠杆菌中的组装辅因子，并检查抗生素对组装过程的影响。我们有 开发了一种新型的同位素脉冲练习测定，该测定能够测量核糖体的结合动力学 蛋白质使用质谱法。我们正在开发两个光子荧光相关显微镜与 实时监测荧光标记的核糖体蛋白的组装。最后，我们正在扩展我们的 对活细菌中同位素脉冲实验的质谱分析，这使我们能够直接 监测核糖体的生物发生。综上所述，这些研究将提供新的机械见解 进入核糖体组装的关键过程。

项目成果

The effect of ribosome assembly cofactors on in vitro 30S subunit reconstitution.

• DOI: 10.1016/j.jmb.2010.02.036
• 发表时间: 2010-04-23
• 期刊: Journal of molecular biology
• 影响因子: 5.6
• 作者: Bunner AE;Nord S;Wikström PM;Williamson JR
• 通讯作者: Williamson JR

Envelope: interactive software for modeling and fitting complex isotope distributions.

• DOI: 10.1186/1471-2105-9-446
• 发表时间: 2008-10-20
• 期刊: BMC bioinformatics
• 影响因子: 3
• 作者: Sykes MT;Williamson JR
• 通讯作者: Williamson JR

Quantitation of the ribosomal protein autoregulatory network using mass spectrometry.

• DOI: 10.1021/ac9028664
• 发表时间: 2010-06-15
• 期刊: ANALYTICAL CHEMISTRY
• 影响因子: 7.4
• 作者: Sykes, Michael T.;Sperling, Edit;Chen, Stephen S.;Williamson, James R.
• 通讯作者: Williamson, James R.

Quantitative proteomic analysis of ribosome assembly and turnover in vivo.

• DOI: 10.1016/j.jmb.2010.08.005
• 发表时间: 2010-10-29
• 期刊: Journal of molecular biology
• 影响因子: 5.6
• 作者: Sykes MT;Shajani Z;Sperling E;Beck AH;Williamson JR
• 通讯作者: Williamson JR

Discovery of a small molecule that inhibits bacterial ribosome biogenesis.

• DOI: 10.7554/elife.03574
• 发表时间: 2014-09-18
• 期刊: eLife
• 影响因子: 7.7
• 作者: Stokes JM;Davis JH;Mangat CS;Williamson JR;Brown ED
• 通讯作者: Brown ED