The Mechanism of tRNA splicing in trypanosomes

在所有生物体中，tRNA通过一系列转录后加工而成熟 在它们参与蛋白质合成之前发生的事件。这些包括末端修剪， 产生正确的5'和3'末端，化学修饰以确保正确的结构 和功能，以及添加氨基酸最终连接的3' CCA尾。在 此外，不同数量的tRNA也含有内含子，这些内含子中断了反密码子 序列，并因此使tRNA无功能。因此，拼接对于 生存能力tRNA内含子的剪接与mRNA剪接的不同之处在于，它涉及几个 蛋白质催化的步骤，包括剪接特异性内切核酸酶的内含子切割， 随后外显子末端成熟并通过三功能连接酶连接（三个连接酶的融合 不同的酶结构域，取决于生物体），最后去除 以前的活动留下的“悬空”磷酸盐。在锥虫中， 含单个内含子的tRNA，tRNATyr，负责解码所有酪氨酸密码子。 到目前为止，人们对任何早期分化中tRNA剪接的机制知之甚少。 原生生物我们的研究已经确定了tRNA每一步的推定成分， 剪接反应，每一个都有不寻常的特征。内含子在几个位置被编辑 并且拼接又需要编辑。剪接核酸内切酶具有亚基 其他系统中缺少一个关键子单元的结构。拼接 连接酶形成更高级的复合物，表明其与其它因子相关。 磷酸转移酶有一个不寻常的核酸外切酶结构域，这表明 在内含子降解中的作用。本提案将侧重于每一种 在T.布鲁塞还将探讨 内含子编辑对剪接特异性的贡献。考虑到T.布氏机制是 充满了独特的功能，成功完成这些研究将产生 关于tRNA剪接的机制和进化的重要基础信息， 了解原生生物学领域。 !

In all organisms, tRNAs are matured by a series of post-transcriptional processing events before they can partake in protein synthesis. These include end trimming to generate the correct 5' and 3' ends, chemical modification to ensure proper structure and function, and addition of a 3' CCA tail where amino acids are ultimately attached. In addition a variable number of tRNAs also contain introns; these interrupt the anticodon sequence and as such render a tRNA non-functional. Therefore, splicing is essential for viability. Splicing of tRNA introns differs from mRNA splicing in that it involves several protein-catalyzed steps, including intron cleavage by a splicing-specific endonuclease, followed by exon-end maturation and ligation by a tri-functional ligase (a fusion of three different enzyme domains, depending on the organism) and lastly removal of a “dangling” phosphate left behind by previous activities. In trypanosomatids, there is a single intron-containing tRNA, tRNATyr, responsible for decoding all the tyrosine codons. To date little is known about the mechanism(s) of tRNA splicing in any early diverging protist. Our studies have have identified putative components for each step of the tRNA splicing reaction, each with unusual features. The intron is edited at several positions and editing is in turn required for splicing. The splicing endonuclease has a sub-unit structure where one of the critical sub-units in other systems is missing. The splicing ligase forms a higher order complex suggesting its association with additional factors. The phosphotransferase has an unusual exonuclease domain suggesting an additional role in intron degradation. This proposal will focus in the characterization of each component of the tRNA splicing pathway in T. brucei and will also explore the contribution of intron editing to splicing specificity. Given that the T. brucei mechanism is peppered with unique features, successful completion of these studies will generate important basic information on the mechanism and evolution of tRNA splicing, a poorly understood area of protist biology. !