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定义tRNA处理的所有生化步骤，并检查其在细胞中的作用。尽管在tRNA上进行了40多年的工作，但我们仍然对这一途径没有完整的了解。当前的工作集中在tRNA修饰上，其中包括大部分未知的生化步骤。酵母细胞中的tRNA分子的数量使任何其他类别的多核苷酸的分子相形见,，则是RRNA的分子的10倍。酵母TRNA中总共有22种不同的修饰，由多个基因产物催化。这些修饰中的许多是高度保守的，生化的未表征，未分配给基因和未知的作用。该领域的最新努力通过遗传，生化和生物信息学方法的结合，导致了许多与修饰有关的基因产物的鉴定。生化基因组学方法的使用导致鉴定了五种涉及四种酵母修饰活动的基因产品。这导致了对许多这些基因产品的特异性，机制和功能的理解的开始，但留下了许多未解决的问题。通过研究几种tRNA修饰酶的新颖而有趣的方面以及鉴定编码修饰活动的新基因，将继续进行tRNA处理的这项工作。提出了四个具体目标。首先，将研究他的Guanyllltransferase的生物化学和生物学。在所有研究的生物中，他的物种与其他TRNA不同，在-1位置额外的G残基，该残基在真核生物中被添加后的转录后添加。已经确定了与tRNA共纯化的两个基因产物。一个是体内活动必不可少的，并且需要。将确定tRNA组成部分的身份和活动，并确定酶识别酶的基础。其次，将检查M1G9甲基转移酶以确定重要的催化和结合残基，因为该酶与此类已知的其他已知酶不同。第三，将确定其他几种修饰酶及其相应的基因。大约17种修饰酶尚未分配给基因。这些修饰在生化上很有趣，并且位于tRNA的重要区域。最后，将在体内研究某些tRNA修饰酶的功能。