The tRNA:m 22 G10 methyltransferase of Pyrococus abyssi (PAB1283, a member of COG1041) catalyzes the N2 , N2 -dimethylation of guanosine at position 10 in tRNA. Boundaries of its THUMP (THioUridine synthases, RNA Methyltransferases and Pseudo-uridine synthases)—containing N-terminal domain [1–152] and C-terminal catalytic domain [157–329] were assessed by trypsin limited proteolysis. An inter-domain flexible region of at least six residues was revealed. The N-terminal domain was then produced as a standalone protein (THUMPa) and further characterized. This autonomously folded unit exhibits very low affinity for tRNA. Using protein fold-recognition (FR) methods, we identified the similarity between THUMPa and a putative RNA-recognition module observed in the crystal structure of another THUMP-containing protein (ThiI thiolase of Bacillus anthracis ). A comparative model of THUMPa structure was generated, which fulfills experimentally defined restraints, i.e. chemical modification of surface exposed residues assessed by mass spectrometry, and identification of an intramolecular disulfide bridge. A model of the whole PAB1283 enzyme docked onto its tRNA Asp substrate suggests that the THUMP module specifically takes support on the co-axially stacked helices of T-arm and acceptor stem of tRNA and, together with the catalytic domain, screw-clamp structured tRNA. We propose that this mode of interactions may be common to other THUMP-containing enzymes that specifically modify nucleotides in the 3D-core of tRNA.