tRNA Splicing

TRNA剪接对于一些真核生物的生存至关重要，如果不是全部的话。TRNA剪接的机制在真核生物中高度保守，在酵母中被最好地理解。从表面上看，tRNA剪接看起来很简单；内切酶切除内含子；连接酶连接两个半分子；2‘-去磷酸化活性去除剪接接头上剩下的2’-磷酸。然而，拼接的机制，特别是最后一步的机制，比人们想象的要复杂得多。剪接连接2‘-磷酸酶的去除不是由磷酸酶催化的，而是由依赖NAD的2’-磷酸特异性磷酸转移酶催化的。像这样的蛋白质从来没有被描述过，但它很可能在一些真核生物中是必不可少的，如果不是所有的话，为了制造有功能的tRNA。此外，来自tRNA剪接连接的磷酸盐最终在NAD或类似于NAD的分子上以环状磷酸盐的形式结束。这种分子以前也没有被描述过。这项建议的目的是确定在tRNA剪接中催化这一不寻常但重要的去磷酸化步骤的蛋白质，并确定这一不寻常的磷酸转移产物的结构(和形成机制)。在酵母中处理新合成的转移RNA(TRNA)的过程对于tRNA在蛋白质合成中的作用是必不可少的。处理的一个重要部分涉及编辑掉链中的特定碱基序列。这项研究涉及一种酶，它可以填补tRNA链上的缝隙(很像中央车厢被移走后火车车厢连接起来的过程)。该酶具有一些新的性质，值得研究以区别于其他连接酶。这些研究产生的信息将是新的和重要的，因为这项研究是原创的。

tRNA splicing is essential for the survival of some, if not all, eukaryotes. The mechanism of tRNA splicing is highly conserved in eukaryotes, and best understood in the yeast Saccharomyces cerevisiae. Superficially, tRNA splicing appears simple; an endonuclease excises the intron; a ligase joins the two half-molecules; and a 2'-dephosphorylating activity removes the 2'-phosphate left at the splice junction. Yet the mechanism of splicing, particularly of the last step, is much more complicated than one would imagine. Removal of the splice junction 2'-phosphatase, rather than being catalyzed by a phosphatase, is catalyzed by an NAD-dependent, 2'-phosphate specific phosphotransferase. A protein like this has never been described, yet it is likely essential in some, if not all, eukaryotes in order to make functional TRNA. Moreover, the phosphate from the splice junction of TRNA ultimately ends up as a cyclic phosphate on NAD, or on a molecule similar to NAD. Such a molecule has also not been previously described. The objects of this proposal are to identify the proteins catalyzing this unusual but important dephosphorylation step in TRNA splicing and to determine the structure (and mechanism of formation) of the unusual phosphotransfer product. %%% The process of processing newly synthesized transfer RNA (TRNA) in yeast is essential for the TRNA to act in the synthesis of a protein. A significant part of the processing involves editing out particular sequences of bases in the strand. This research involves an enzyme which closes a gap in the TRNA strand (much like the process by which train cars are linked up after the cars in the center have been removed). The enzyme has some novel properties which merit investigation to distinguish it from other ligating enzymes . The information resulting from these studies will be new and important since the research is original.