Enzymatic basis for cytidine to uridine editing on archaeal tRNAs

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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.