Enzymatic basis for cytidine to uridine editing on archaeal tRNAs

到目前为止，在真核细胞器tRNA和布氏锥虫细胞质中的tRNAs中已经描述了tRNA中胞苷（C）到尿苷（U）的转录后RNA编辑。然而，C到U编辑过程在生物化学上知之甚少，涉及的酶也未知。在本申请中，将研究Methanopyrus kandleri tRNA的相应脱氨基作用。首先，将在M的无细胞提取物中建立酶测定。kandleri鉴定作用于tRNA的推定胞苷脱氨酶活性。在第二种互补方法中，将表征在生物信息学方法中鉴定的来自开放阅读框MK 0935的候选蛋白。它是一种具有脱氨酶基序的孤儿蛋白，此外还含有一个THUMP结构域;已知该蛋白质结构域参与tRNA结合。使用体外转录底物tRNA对重组MK 0935蛋白进行活性测定。随后将对该蛋白进行生化表征，探索tRNA结合和催化特征。结果应该允许获得的整体脱氨过程的图片，其贡献的功能翻译机器在这个超嗜热古生物。

The posttranscriptional RNA editing of cytidine (C) to uridine (U) in tRNAs has so far been described in eukaryotic organellar tRNAs and also for tRNAs in the Trypanosoma brucei cytoplasm. However, the C to U editing progress is biochemically poorly understood and the enzymes involved are unknown. In this application, the corresponding deamination of Methanopyrus kandleri tRNAs will be investigated. First, an enzyme assay will be established in cell-free extracts of M. kandleri to identify the putative cytidine deaminase activity that acts on tRNA. In a second complementary approach, the candidate protein from open reading frame MK0935, which was identified in a bioinformatics approach, will be characterized. It is an orphan protein with deaminase motifs and, in addition, contains a THUMP domain; this protein domain is known to participate in tRNA binding. The recombinant MK0935 protein will be subjected to activity assays using in vitro transcribed substrate tRNA. A biochemical characterization of the protein will follow, exploring tRNA binding and catalytic features. The results should allow to gain a picture of the overall deamination process and its contribution to a functional translation machinery in this hyperthermophile archaeaon.