A novel nucleotide incorporation activity implicated in the editing of mitochondrial transfer RNAs in Acanthamoeba castellanii

In Acanthamoeba castellanii, most of the mtDNA-encoded tRNAs are edited by a process that replaces one or more: of the first three nucleotides at thei r 5' ends. As a result, base pairing potential is restored at acceptor stem positions (1:72, 2:71, and/or 3:70, in standard tRNA nomenclature) that ar e mismatched according to the corresponding tRNA gene sequence. Here we des cribe a novel nucleotide incorporation activity, partially purified from A. castellanii mitochondria, that has properties implicating it in mitochondr ial tRNA editing in this organism. This activity is able to replace nucleot ides at the first three positions of a tRNA (positions 1, 2, and 3), matchi ng the newly incorporated residues through canonical base pairing to the re spective partner nucleotide in the 3' half of the acceptor stem. Labeling e xperiments with natural (Escherichia coli tRNA(Tyr)) and synthetic (run-off transcripts corresponding to A. castellanii mitochondrial tRNA(Leu1)) subs trates suggest that the nucleotide incorporation activity consists of at le ast two components, a 5' exonuclease or endonuclease and a template-directe d 3'-to-5' nucleotidyltransferase. The nucreotidyltransferase component dis plays an ATP requirement and generates 5' pppN... termini in vitro. The dev elopment of an accurate and efficient in vitro system opens the way for det ailed studies of the biochemical properties of this novel activity and its relationship to mitochondrial tRNA editing in A. castellanii. In addition, the system will allow delineation of the structural features In a tRNA that identify it as a substrate for the labeling activity.