IDENTIFICATION OF MODIFIED NUCLEOSIDES IN RIBOSOMAL RNA

核糖体 RNA 中修饰核苷的鉴定

• 批准号: 6351287
• 负责人: PATRICK A LIMBACH
• 金额: $ 13.04万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 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 2002-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6351287
• 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， rRNA位于核糖体的功能位点附近。但 这些修饰的识别仍然是有问题的， 功能仍然未知。这项研究的长期目标是 表征和理解这些修饰的功能， 特别是关于它们在蛋白质合成中的作用， 开发用于其结构表征的改进方法。 具体而言，将使用基于质谱的方案来 确定转录后的序列位置和身份 rRNA中的修饰核苷。用内切酶选择性切割rRNA， 外切核酸酶，然后质谱分析这些消化 产品将允许后的身份和序列定位， 转录修饰有待确定。该确定 通过比较实验测量的质量值和预测的质量值， 从基因序列中。修饰位点（假尿苷除外） 表现为测量的分子量的增加。的 假尿苷的鉴定是唯一的沉默修饰， 使用特定的化学反应进行， 假尿苷残基允许使用质谱鉴定它们 光谱法这些方法的初步开发将在 以大肠杆菌16S和23S为模型系统。的方法 这里开发的方法适用于广泛的核酸（例如， snRNA、mRNA、tRNA）。本研究 将提供一种快速、准确的序列分析方法， rRNA，并将允许更详细的研究功能的重要性， 蛋白质合成中的转录后修饰。