RUI: Identification of tRNA Maturation Proteins in Yeast

Wolfe转移RNA作为前体在细胞核中合成，在细胞核中经历一系列成熟步骤，包括5'和3'序列的切割、碱基修饰、间插序列的去除和寡核苷酸CCA至3'末端的添加。然后转移RNA被输出到胞质溶胶中，在胞质溶胶中另外的碱基被修饰并且3'CCA末端被修复。在线粒体中，前体tRNA经历类似的成熟过程。 细胞核和线粒体中成熟途径的许多成分尚未被鉴定。 这项研究将确定新的组成部分的tRNA成熟途径，检查这些蛋白质的组织在一个假定的tRNA成熟复合物，并研究蛋白质的相互作用，重要的有效成熟的tRNA。 交联和免疫沉淀实验将利用已知的成熟蛋白作为锚来分离和鉴定tRNA成熟途径的其他组分。 由于Trm1p、Mod5p和Cca1p在核和线粒体前体tRNA的成熟中起作用，因此它们将被用作锚点，以生物化学方式从两种细胞器中分离出化学量的tRNA成熟复合物。 交联技术将允许分离与前体tRNA相互作用的沿着锚蛋白的蛋白质。将通过肽序列分析鉴定这些复合物的组分。然后，肽序列信息将用于搜索酵母基因组数据库中的相应基因。将采用双杂交选择来确定tRNA成熟复合物中新鉴定的蛋白质之间的相互作用模式。将进行遗传实验，以探索Ccalp在tRNA代谢中的核心作用。 ATP（CTP）：tRNA核苷酸转移酶（Ccalp）是迄今为止唯一被表征的tRNA成熟酶，其对于所有细胞tRNA的成熟是必需的。Ccalp定位于细胞核、线粒体和胞质溶胶，在那里它催化寡核苷酸CCA添加到细胞核和线粒体前体tRNA的3'末端。在细胞质中，Cca1p修复CCA末端缩短的tRNA。将进行遗传合成致死筛选，以鉴定在特定细胞区室中发生但可能无法通过生化方法检测到的基本Ccalp相互作用。从这些生物化学和遗传学方法获得的实验数据将产生新的信息，关于tRNA成熟蛋白的身份，tRNA成熟复合物的组织和细胞中tRNA代谢所必需的蛋白质相互作用。 转移RNA作为前体在细胞核中合成，在那里它经历一系列成熟步骤。 在线粒体中，前体tRNA经历类似的成熟过程。 细胞核和线粒体中成熟途径的许多成分尚未被鉴定。 从这些生物化学和遗传学实验中获得的数据将产生关于鉴定tRNA成熟蛋白、tRNA成熟复合物的组织以及细胞中tRNA代谢所必需的蛋白质相互作用的新信息。

9723045 Wolfe Transfer RNA is synthesized as a precursor in the nucleus where it undergoes a series of maturation steps that include the cleavage of 5' and 3' sequences, base modifications, the removal of intervening sequences, and the addition of the oligonucleotide CCA to the 3' terminus. Transfer RNA is then exported to the cytosol where additional bases are modified and the 3'CCA terminus is repaired. In the mitochondria, precursor tRNA undergoes a similar maturation process. Many of the components of the maturation pathway in both the nucleus and mitchondria have not yet been identified. This research will identify new components of the tRNA maturation pathway, examine the organization of these proteins in a putative tRNA maturation complex, and to study protein interactions important to efficient maturation of tRNA. Crosslinking and immunoprecipitation experiments will utilize known maturation proteins as anchors to isolate and identify other components of the tRNA maturation pathway. Since Trm1p, Mod5p and Cca1p function in the maturation of nuclear and mitochondrial pre-tRNA they will be utilized as anchors to biochemically isolate chemical amounts of tRNA maturation complexes from both organelles. Crosslinking techniques will allow the isolation of proteins that along with the anchor protein interact with pre-tRNA. Components of these complexes will be identified by peptide sequence analysis. Peptide sequence information will then be used to search the yeast genomic data base for the corresponding gene. A two hybrid selection will be employed to determine the pattern of interactions between the newly identified proteins in the tRNA maturation complex. Genetic experiments will be conducted that exploit the central role of Ccalp in tRNA metabolism. ATP(CTP): tRNA nucleotidyhxansferase (Ccalp) is the only tRNA maturation enzyme characterized thus far that is essential for maturation of all cellular tRNAs. Ccalp localizes to the nucleus, mitochondria, and cytosol where it catalyzes the addition of the oligonudeotides CCA to the 3' end of nuclear and mitochondrial precursor tRNA. In the cytosol Cca1p repairs CCA end-shortened tRNAs. A genetic synthetic lethal screen will be carried out to identify essential Ccalp interactions that occur in a specific cellular compartment but may not be detected by biochemical means. Experimental data obtained from these biochemical and genetic approaches will yield new information concerning the identity of tRNA maturation proteins, the organization of tRNA maturation complexes and the protein interactions essential to tRNA metabolism in the cell. Transfer RNA is synthesized as a precursor in the nucleus where it undergoes a series of maturation steps. In the mitchondria, precursor tRNA undergoes a similar maturation process. Many of the components of the maturation pathway in both the nucleus and mitchondria have not yet been identified. Data obtained from these biochemical and genetic experiments will yield new information concerning the identify of tRNA maturation proteins, the organization of tRNA maturation complexes and the protein interactions essential to tRNA metabolism in the cell.