title: 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.
authors: CEA VALRHO, DSV-DIEP-SBTN, Service de Biochimie post-génomique & Toxicologie Nucléaire, F-30207 Bagnols-sur-Cèze, France.
authors: Guillaume
authors: G
authors: Gabant
authors: Sylvie
authors: S
authors: Auxilien
authors: Irina
authors: I
authors: Tuszynska
authors: Marie
authors: M
authors: Locard
authors: Michal J
authors: MJ
authors: Gajda
authors: Guylaine
authors: G
authors: Chaussinand
authors: Bernard
authors: B
authors: Fernandez
authors: Alain
authors: A
authors: Dedieu
authors: Henri
authors: H
authors: Grosjean
authors: Béatrice
authors: B
authors: Golinelli-Pimpaneau
authors: Janusz M
authors: JM
authors: Bujnicki
authors: Jean
authors: J
authors: Armengaud