THUMP from archaeal tRNA: m22G10 methyltransferase, a genuine autonomously folding domain

The tRNA:m(2) (2)G10 methyltransferase of Pyrococus abyssi (PAB1283, a member of COG1041) catalyzes the N-2,N-2-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 (THUMP alpha) and further characterized. This autonomously folded unit exhibits very low affinity for tRNA. Using protein fold-recognition (FR) methods, we identified the similarity between THUMP alpha 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 THUMP alpha 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.