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

Abstract : The tRNA:m2(2)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 (THUMPalpha) and further characterized. This autonomously folded unit exhibits very low affinity for tRNA. Using protein fold-recognition (FR) methods, we identified the similarity between THUMPalpha 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 THUMPalpha 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.
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Submitted on : Friday, May 17, 2019 - 4:46:50 PM
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Guillaume Gabant, Sylvie Auxilien, Irina Tuszynska, Marie Locard, Michal Gajda, et al.. THUMP from archaeal tRNA:m22G10 methyltransferase, a genuine autonomously folding domain. Nucleic Acids Research, Oxford University Press, 2006, 34 (9), pp.2483-2494. ⟨10.1093/nar/gkl145⟩. ⟨hal-02132929⟩



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