tRNA Splicing in Yeast

酿酒酵母中tRNA分子的内含子是如何剪接的？TRNA剪接在酵母中是必不可少的。酵母中tRNA剪接的研究是在各种生物体中剪接的范例，因为剪接机制在这些生物体中是高度保守的。剪接包括由内切酶切除内含子，通过连接酶连接所产生的半分子，以及去除留在剪接连接处的2‘-磷酸。由于这种磷酸盐是tRNA反密码子的一个碱基3‘，因此它的去除可能是产生功能tRNA所必需的。去除这种2‘-磷酸是这项提议的主题。PI最近从酵母中鉴定并部分纯化了一种蛋白质，该蛋白质可以有效地从连接的tRNA中去除剪接结磷酸盐。这种蛋白质是独一无二的：它是2‘-磷酸化底物所特有的，需要NAD+，并作为一种磷酸转移酶。它的活性似乎也受到携带tpd1突变的细胞的影响。他建议确定NAD+如何用于连接的tRNA的去磷酸化，并确定参与这一反应的蛋白质。他还建议确定TPD1蛋白如何影响细胞中连接的tRNA的去磷酸化，并研究2‘-磷酸转移酶与tRNA剪接机制的其他组件的相互作用。

How are introns spliced from tRNA molecules in the yeast Saccharomyces cerevisiae? tRNA splicing is essential in yeast. Study of tRNA splicing in yeast is a paradigm for splicing in a wide variety of organisms since the mechanism of splicing is highly conserved in these organisms. Splicing involves excision of the intron by an endonuclease, ligation of the resultant half- molecules by a ligase, and removal of a 2'-phosphate left at the splice junction. Since this phosphate is one base 3' of the tRNA anticodon its removal is likely necessary to generate functional tRNA. Removal of this 2'-phosphate is the subject of this proposal. The PI has recently identified and partially purified a protein from yeast that can efficiently remove the splice junction phosphate from ligated tRNA. This protein is unique: it is specific for 2'-phosphorylated substrates, requires NAD+ and acts as a phosphotransferase. Its activity also appears to be affected by cells carrying a tpd1 mutation. He proposes to determine how NAD+ is used in dephosphorylation of ligated tRNA and to identify the proteins involved in this reaction. He also proposes to determine how TPD1 protein affects dephosphorylation of ligated tRNA in the cell, and to study the interaction of the 2'-phosphotransferase with other components of the tRNA splicing machinery.