Mass Spectrometry of Ribosomal RNA:Protein Interactions

核糖体 RNA:蛋白质相互作用的质谱分析

• 批准号: 7879682
• 负责人: PATRICK A LIMBACH
• 金额: $ 10.23万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-17 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7879682
• 关键词: Affinity Chromatography; Amaze; Antibiotics; Antibodies; Area; Cells; Complex; Development; Eukaryota; Funding; Future; Goals; Knowledge; Maps; Mass Spectrum Analysis; Methods; Modification; Molecular; Nature; Nucleic Acids; Nucleosides; Organism; Pattern; Proteins; Proteolysis; Proteomics; Pseudouridine; RNA; RNA-Protein Interaction; Research; Research Personnel; Resolution; Ribonuclease H; Ribonucleoproteins; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Role; Site; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Speed; Structure; System; Technology; Translations; Work; base; cell growth; crosslink; improved; interest; liquid chromatography mass spectrometry; method development; novel strategies; protein complex; research study

DESCRIPTION: (provided by applicant): Posttranscriptional modifications in ribosomal ribonucleic acids (rRNAs) are difficult to identify and their function is unknown. Our knowledge of the functional role of these posttranscriptional modifications is limited largely by the lack of methods for their routine identification. The long-term goal of our research has been and continues to be to develop appropriate mass spectrometric approaches to characterize the structure of the ribosome in terms of RNA. The goal of this funding period will be to extend our previous improvements in mass spectrometric methods for identifying posttranscriptional modifications in rRNA in order to permit the structural interactions between (modified) nucleosides in rRNA and ribosomal proteins to be characterized. We will develop new, improved mass spectrometry (MS) methods to characterize ribonucleoprotein (RNP) interactions within the ribosome. We will use matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to identify cross-links between RNA and ribosomal proteins. We will extend our current developments using RNase H to digest rRNAs to focus on particular regions of the ribosome as a means of simplifying the determination of protein:nucleic acid cross-links. In particular, we will use this new approach to focus on areas of rRNA known to contain posttranscriptionally modified nucleosides to ascertain the structural significance of such modifications. We will use limited proteolysis with MALDI-MS to characterize ribosome organization, topology and proteins suspected of directly interacting with rRNA. Limited proteolysis, cross-linking and analysis by MALDI-MS and liquid chromatography mass spectrometry will be used to identify sites of interaction between ribosomal proteins and rRNAs suspected of being important in ribosome assembly. As pseudouridine is the most common posttranscriptionally modified nucleoside, we propose to develop an improved method for identifying this modification and analyzing its role in rRNA:rRNA and rRNA:protein interactions. Furthermore, we will apply advances in method developments in proteomics to initiate studies aimed at characterizing the structural interactions of bacterial ribosomes as a function of cell growth conditions. These latter studies will open up new avenues of research in characterizing the dynamic nature of the ribosome. In addition, this research will facilitate future studies that will help determine how ribosomes can carry.out the task of translation with amazing speed and accuracy.

描述：（由申请人提供）：核糖体核糖核酸（rRNA）的转录后修饰很难鉴定，并且其功能未知。我们对这些转录后修饰的功能作用的了解很大程度上受到缺乏常规识别方法的限制。我们研究的长期目标一直是并将继续是开发适当的质谱方法来表征核糖体的 RNA 结构。本资助期的目标是扩展我们之前在鉴定 rRNA 转录后修饰的质谱方法方面的改进，以便能够表征 rRNA 中（修饰的）核苷和核糖体蛋白之间的结构相互作用。我们将开发新的、改进的质谱（MS）方法来表征核糖核蛋白（RNP）在核糖体内的相互作用。我们将使用基质辅助激光解吸/电离质谱 (MALDI-MS) 来识别 RNA 和核糖体蛋白之间的交联。我们将利用 RNase H 消化 rRNA 来扩展我们目前的开发，重点关注核糖体的特定区域，作为简化蛋白质：核酸交联测定的手段。特别是，我们将使用这种新方法关注已知含有转录后修饰核苷的 rRNA 区域，以确定此类修饰的结构意义。我们将使用 MALDI-MS 进行有限的蛋白水解来表征核糖体组织、拓扑结构以及怀疑与 rRNA 直接相互作用的蛋白质。 MALDI-MS 和液相色谱质谱法的有限蛋白水解、交联和分析将用于识别核糖体蛋白和疑似在核糖体组装中重要的 rRNA 之间的相互作用位点。由于假尿苷是最常见的转录后修饰核苷，我们建议开发一种改进的方法来识别这种修饰并分析其在 rRNA:rRNA 和 rRNA:蛋白质相互作用中的作用。此外，我们将应用蛋白质组学方法开发的进展来启动研究，旨在表征细菌核糖体的结构相互作用作为细胞生长条件的函数。后面的这些研究将为表征核糖体的动态性质开辟新的研究途径。此外，这项研究将促进未来的研究，帮助确定核糖体如何以惊人的速度和准确性执行翻译任务。