THUMP from archaeal tRNA:m22G10 methyltransferase, a genuine autonomously folding domain |
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| Authors: | Gabant Guillaume Auxilien Sylvie Tuszynska Irina Locard Marie Gajda Michal J Chaussinand Guylaine Fernandez Bernard Dedieu Alain Grosjean Henri Golinelli-Pimpaneau Béatrice Bujnicki Janusz M Armengaud Jean |
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| Affiliation: | Guillaume Gabant, Sylvie Auxilien, Irina Tuszynska, Marie Locard, Michal J. Gajda, Guylaine Chaussinand, Bernard Fernandez, Alain Dedieu, Henri Grosjean, Béatrice Golinelli-Pimpaneau, Janusz M. Bujnicki, and Jean Armengaud |
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| Abstract: | The tRNA:m22G10 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 (THUMPα) and further characterized. This autonomously folded unit exhibits very low affinity for tRNA. Using protein fold-recognition (FR) methods, we identified the similarity between THUMPα 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 THUMPα 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 tRNAAsp 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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