tRNA Processing

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的研究已经有40多年了，但我们仍然没有对这一途径有一个完整的了解。目前的工作集中在tRNA修饰上，这构成了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别的多核苷酸相形见绌，其分子数量是rRNA的10倍。酵母tRNAs共有22种不同的修饰，由多个基因产物催化。许多这些修饰是高度保守的，没有生化特征，没有分配给基因，作用未知。最近在这一领域的努力导致了使用遗传、生化和生物信息学方法的组合来鉴定一些涉及修饰的基因产物。利用生化基因组学方法，鉴定出了与四种酵母修饰活动有关的五种基因产物。这导致了对许多这些基因产物的特异性、机制和功能的初步了解，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，并通过识别编码修饰活性的新基因来继续。提出了四个具体目标。首先，对tRNA-His鸟苷酸转移酶的生物化学和生物学进行了研究。来自所有被研究生物的他的物种与其他tRNA的不同之处在于在-1位有额外的G残基，这在真核生物中是在转录后添加的。已经鉴定了两种与tRNA共同纯化鸟苷酰转移酶活性的基因产物；其中一种是体内活性所必需的。将确定tRNA组份的特性和活性，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，因为该酶不同于其他已知的此类酶。第三，将确定其他几种修饰酶及其相应的基因。大约有17种修饰酶还没有被分配给基因。许多这些修饰在生物化学上很有趣，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。

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.