Mass Spectrometry of Ribosomal RNA:Protein Interactions

描述：(申请人提供)：核糖体核糖核酸(RRNA)的转录后修饰很难识别，其功能尚不清楚。我们对这些转录后修饰的功能作用的了解在很大程度上受到缺乏常规鉴定方法的限制。我们研究的长期目标一直是并将继续发展适当的质谱学方法来表征核糖体的RNA结构。这一资助期的目标将是扩大我们以前在鉴定rRNA转录后修饰的质谱学方法方面的改进，以便能够表征rRNA和核糖体蛋白中(修饰的)核苷之间的结构相互作用。我们将开发新的、改进的质谱学(MS)方法来表征核糖体中的核糖核蛋白(RNP)相互作用。我们将使用基质辅助激光解吸/电离质谱仪(MALDI-MS)来鉴定RNA和核糖体蛋白之间的交联键。我们将扩展我们目前的开发，使用RNaseH来消化rRNA，将重点放在核糖体的特定区域，作为一种简化蛋白质测定的手段：核酸交联链。特别是，我们将使用这种新的方法来关注已知的含有转录后修饰核苷的rRNA区域，以确定这种修饰的结构意义。我们将使用MALDI-MS的有限蛋白质分解来表征核糖体的组织、拓扑结构和怀疑与rRNA直接相互作用的蛋白质。有限的蛋白质分解、MALDI-MS和液质联用分析将被用来确定核糖体蛋白和怀疑在核糖体组装中重要的rRNA之间的相互作用部位。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鉴定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发展的进展来启动旨在表征细菌核糖体作为细胞生长条件函数的结构相互作用的研究。这些后面的研究将在表征核糖体的动态性质方面开辟新的研究途径。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完成翻译任务。

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.