tRNA Processing

tRNA加工

• 批准号: 6829734
• 负责人: Eric M. Phizicky
• 金额: $ 29.93万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6829734
• 关键词: Escherichia coli; SDS polyacrylamide gel electrophoresis; biochemistry; enzyme activity; guanosine triphosphate; mass spectrometry; methyltransferase; polynucleotides; protein purification; pyrophosphatase; transfer RNA; yeasts

The long term goal of this project is to define all the biochemical steps of tRNA processing and to examine their roles in the cell. Despite more than 40 years of work on tRNA, we still do not have a complete picture of this pathway. Current work is focused on tRNA modifications, which comprise the bulk of unknown biochemical steps. The number of tRNA molecules in the yeast cell dwarfs that of any other class of polynucleotide, accounting for 10 times more molecules than rRNA. There are a total of 22 different modifications in yeast tRNAs, catalyzed by multiple gene products. Many of these modifications are highly conserved, biochemically uncharacterized, unassigned to genes, and of unknown role. Recent efforts in the field have led to the identification of a number of gene products involved in modification, using a combination of genetic, biochemical, and bioinformatic methods. Use of a biochemical genomics approach led to the identification of five gene products involved in four yeast modification activities. This has led to the beginnings of an understanding of the specificity, mechanism and function of many of these gene products, but leaves many unanswered questions. This work on tRNA processing will be continued by studying novel and interesting aspects of several tRNA modification enzymes, and by identifying new genes encoding modification activities. Four specific aims are proposed. First, the biochemistry and biology of tRNA His guanylyltransferase will be studied. tRNA His species from all studied organisms are different from other tRNAs in having an extra G residue at the -1 position, which is added post-transcriptionally in eukaryotes. Two gene products that co-purify with tRNA His guanylyltransferase activity have been identified; one is essential and required for activity in vivo. The identities and activities of components of tRNA His guanylyltransferase will be determined, as well as the basis for tRNA recognition by the enzyme. Second, m1G9 methyltransferase will be examined to determine important catalytic and binding residues, because this enzyme is different from other known enzymes of this type. Third, several other modification enzymes and their corresponding genes will be identified. Some 17 modification enzymes have not yet been assigned to genes. Many of these modifications are biochemically interesting and are located in important regions of the tRNA. Finally, the function of some tRNA modification enzymes will be studied in vivo.

该项目的长期目标是定义 tRNA 加工的所有生化步骤并检查它们在细胞中的作用。尽管对 tRNA 的研究已有 40 多年的历史，但我们仍然没有全面了解该通路。目前的工作重点是 tRNA 修饰，其中包含大量未知的生化步骤。酵母细胞中 tRNA 分子的数量使任何其他类别的多核苷酸相形见绌，其分子数量是 rRNA 的 10 倍。酵母 tRNA 共有 22 种不同的修饰，由多种基因产物催化。许多这些修饰是高度保守的、生化上未表征的、未分配给基因的且作用未知。该领域最近的努力已经通过结合遗传、生物化学和生物信息学方法鉴定了许多参与修饰的基因产物。使用生化基因组学方法鉴定了涉及四种酵母修饰活动的五种基因产物。这使得人们开始了解许多这些基因产物的特异性、机制和功能，但留下了许多悬而未决的问题。这项关于 tRNA 加工的工作将通过研究几种 tRNA 修饰酶的新颖和有趣的方面以及识别编码修饰活性的新基因来继续进行。提出了四个具体目标。首先，将研究tRNA组氨酸鸟苷基转移酶的生物化学和生物学。来自所有研究生物体的 tRNA His 物种与其他 tRNA 的不同之处在于，在 -1 位点有一个额外的 G 残基，该残基是在真核生物中转录后添加的。已鉴定出两种与 tRNA His 鸟苷基转移酶活性共纯化的基因产物；其中之一对于体内活动是必需的和必需的。将确定 tRNA His 鸟苷基转移酶组分的身份和活性，以及​​该酶识别 tRNA 的基础。其次，将检查 m1G9 甲基转移酶以确定重要的催化和结合残基，因为该酶不同于该类型的其他已知酶。第三，将鉴定其他几种修饰酶及其相应的基因。大约 17 种修饰酶尚未被分配给基因。其中许多修饰在生物化学上都很有趣，并且位于 tRNA 的重要区域。最后，一些tRNA修饰酶的功能将在体内进行研究。