IDENTIFICATION OF MODIFIED NUCLEOSIDES IN RIBOSOMAL RNA

核糖体 RNA 中修饰核苷的鉴定

• 批准号: 6618494
• 负责人: PATRICK A LIMBACH
• 金额: $ 1.1万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6618494
• 关键词: Escherichia coli; bacterial RNA; bacterial genetics; enzyme activity; exonuclease; high performance liquid chromatography; matrix assisted laser desorption ionization; method development; nucleic acid probes; nucleic acid sequence; oligonucleotides; point mutation; posttranscriptional RNA processing; ribonuclease H; ribosomal RNA; uracil nucleoside

The locations of the majority of post-transcriptional modifications in rRNA are near the functional site of the ribosome. However, the identification of these modifications remains problematic and their function remains unknown. The long term goal of this research is the characterize and understanding the function of these modifications, especially concerning their role in protein synthesis, is the development of improved methods for their structural characterization. Specifically, a mass spectroscopy-based protocol will be used to determine the sequence location and identity of post-transcriptionally modified nucleosides in rRNA. Selective cleavage of rRNA with endo- and exonucleases followed by mass spectrometric analysis of these digestion products will permit the identity and sequence location of post- transcriptional modifications to be determined. This determination is made by comparing experimentally measured mass values to that predicted from the gene sequence. Sites of modification (except for pseudouridine) are manifested as an increase in the measured molecular weight. The identification of pseudouridine, the only silent modification, will be performed using specific chemical reactions that shift the mass of all pseudouridine residues permitting their identification using mass spectrometry. The initial development of these methods will be performed with Escherichia coli 16S and 23S as the model systems. The methods developed here are applicable to a wide range of nucleic acids (e.g., snRNA, mRNA, tRNA) which contain modified nucleosides. This research will provide a rapid and accurate method for the sequence analysis of rRNA and will permit more detailed studies on the functional importance of post-transcriptional modifications in protein synthesis.

大多数转录后修饰的位置在 RRNA位于核糖体的功能部位附近。然而， 对这些修改的识别仍然存在问题，他们的 功能仍不清楚。这项研究的长期目标是 描述和理解这些修改的功能， 尤其是关于它们在蛋白质合成中的作用，是 为它们的结构表征开发改进的方法。 具体地说，基于质谱学的协议将用于 转录后确定序列的位置和同源性 RRNA中的修饰核苷。内切酶与rRNA的选择性切割 核酸外切酶和这些酶的质谱分析 产品将允许邮寄的身份和顺序位置- 转录修饰有待确定。这一决心是 将实验测量的质量值与预测的质量值进行比较 从基因序列中。修饰部位(假尿苷除外) 表现为测得的相对分子质量增加。这个 伪尿嘧啶的鉴定，唯一的沉默修改，将是 利用特定的化学反应将所有物质的质量 假尿苷残基允许使用质量法进行鉴定 光谱分析。将进行这些方法的初步开发 以大肠杆菌16S和23S为模型系统。这些方法 在此开发的可适用于广泛的核酸(例如， 含有修饰的核苷的SnRNA、mRNA、tRNA)。这项研究 将为序列分析提供一种快速、准确的方法 RRNA，并将允许对功能重要性进行更详细的研究 蛋白质合成中的转录后修饰。