RUI: Identification of tRNA Maturation Proteins in Yeast

9723045沃尔夫转移核糖核酸是在细胞核中作为前体合成的，在那里它经历了一系列成熟步骤，包括5‘和3’序列的裂解、碱基修饰、中间序列的去除和在3‘端添加寡核苷酸CCA。然后，转移RNA输出到细胞质，在那里额外的碱基被修饰，3‘CCA末端被修复。在线粒体中，前体tRNA经历了类似的成熟过程。细胞核和线粒体中成熟途径的许多成分尚未确定。本研究将鉴定tRNA成熟途径的新成分，研究这些蛋白质在假定的tRNA成熟复合体中的组织结构，并研究对tRNA有效成熟至关重要的蛋白质相互作用。交联和免疫沉淀实验将利用已知的成熟蛋白作为锚，分离和鉴定tRNA成熟途径的其他成分。由于Trm1p、Mod5p和Cca1p在核和线粒体前tRNA的成熟过程中起作用，它们将被用作锚，从这两个细胞器中以生物化学的方式分离化学含量的tRNA成熟复合体。交联化技术将允许分离与锚定蛋白一起与前tRNA相互作用的蛋白质。这些复合体的组成将通过多肽序列分析进行鉴定。然后，肽序列信息将被用于在酵母基因组数据库中搜索相应的基因。将采用双杂交选择来确定tRNA成熟复合体中新发现的蛋白质之间的相互作用模式。将进行基因实验，利用Ccalp在tRNA新陈代谢中的核心作用。三磷酸腺苷(CTP)：tRNA核苷酸转移酶(Ccalp)是迄今为止唯一的tRNA成熟酶，对所有细胞tRNA的成熟都是必不可少的。Ccalp定位于细胞核、线粒体和胞浆，在那里它催化将寡核苷酸CCA加到核和线粒体前体tRNA的3‘端。在胞浆中，Cca1p修复CCA末端缩短的tRNA。将进行遗传合成致死筛选，以确定发生在特定细胞室但可能无法通过生化手段检测到的基本Ccalp相互作用。从这些生化和遗传方法获得的实验数据将产生关于tRNA成熟蛋白的身份、tRNA成熟复合体的组织以及tRNA在细胞中代谢所必需的蛋白质相互作用的新信息。转移RNA作为前体在细胞核中合成，在那里它经历了一系列成熟步骤。在线粒体中，前体tRNA经历了类似的成熟过程。细胞核和线粒体中成熟途径的许多成分尚未确定。从这些生化和遗传学实验中获得的数据将产生关于tRNA成熟蛋白的鉴定、tRNA成熟复合体的组织以及tRNA在细胞中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.