tRNA Processing

tRNA处理

• 批准号: 6986065
• 负责人: Eric M. Phizicky
• 金额: $ 29.22万
• 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/6986065
• 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确定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的研究已经超过40年，但我们仍然没有对这一途径的完整了解。目前的工作集中在tRNA修饰上，这包括大部分未知的生化步骤。酵母细胞中tRNA分子的数量使任何其他类型的多核苷酸相形见绌，比rRNA分子多10倍。在酵母tRNA中总共有22种不同的修饰，由多种基因产物催化。这些修饰中的许多是高度保守的，生物化学上未表征的，未分配给基因的，以及未知的作用。最近在该领域的努力已经导致了一些基因产物的鉴定涉及修改，使用遗传，生物化学和生物信息学方法的组合。使用的生化基因组学方法，导致五个基因产物参与四个酵母修饰活动的鉴定。这导致了对许多这些基因产物的特异性、机制和功能的理解的开始，但留下了许多未解答的问题。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 His鸟苷酰转移酶的生物化学和生物学。来自所有研究的生物体的tRNA His种类与其他tRNA不同，在-1位具有额外的G残基，其在真核生物中转录后添加。已经鉴定了两种与tRNA His鸟苷酰转移酶活性共纯化的基因产物;一种是必需的，并且是体内活性所需的。将确定tRNA His鸟苷酰转移酶组分的身份和活性，以及该酶识别tRNA的基础。第二，将检查m1 G9甲基转移酶以确定重要的催化和结合残基，因为这种酶不同于这种类型的其他已知酶。第三，将鉴定几种其他修饰酶及其相应的基因。大约有17种修饰酶尚未被指定为基因。这些修饰中的许多是生物化学上有趣的，并且位于tRNA的重要区域。最后，将在体内研究一些tRNA修饰酶的功能。