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

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.

The open reading frame TTC1157 of Thermus thermophilus HB27 encodes the methyltransferase forming N2-methylguanosine at position 6 in tRNA

“…Our results indicate that the THUMP domain alone is not sufficient for tRNA binding. A similar observation was made by Gabant et al (2006) on the THUMP domain of archaeal TrmG10. Also, the individual MTase domain does not show any tRNA binding.…”

“…The Trm1 enzyme bears a putative helix-turn-helix motif at its C terminus, while the TrmG10 protein contains an N-terminal THUMP domain (Armengaud et al 2004). The THUMP domain, present in thiouridine synthases, RNA MTases, and pseudouridine synthases (Aravind and Koonin 2001), was proposed to interact with a specific region of the tRNA, thereby targeting the catalytic domain of the enzyme toward a particular region of the tRNA structure (Gabant et al 2006).…”

Deduced RNA binding mechanism of ThiI based on structural and binding analyses of a minimal RNA ligand

“…To date, there have been no reports confirming the interaction between the THUMP domain and RNA, although it is found in several RNA modification enzymes and is therefore likely to serve as a general module for interaction with RNA (Aravind and Koonin 2001;Gabant et al 2006). The present study is the first to confirm this.…”

“…Spectral changes in this region were reported to indicate conformational changes of nucleic acid molecules (Connor et al 1994;Gray et al 1995;Nejedly et al 2005;Saito et al 2008). Gabant et al (2006) reported for ThiI and other tRNA modification enzymes that some conformational Phe and (B) TPHE39A: (lanes 1,5,9) 100 pmol protein; (lanes 2,6,10) 100 pmol RNA; (lanes 3,7,11) 100 pmol protein + 100 pmol RNA; and (lanes 4,9,12) 200 pmol protein + 100 pmol RNA. RT ln K A = DH À TDS.…”

“…As the THUMP domain is common to several RNA binding proteins, it likely acts as an RNA recognition motif (Aravind and Koonin 2001;Gabant et al 2006). The crystal structures of ThiI from Pyrococcus horikoshii and Bacillus anthracis, which did not contain RLD, revealed an elongated structure in which the THUMP domain and the PP-loop domain were separated, with NFLD located between them (Waterman et al 2006).…”