THUMP from archaeal tRNA:m(2)(2)G10 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 (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 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

Structural requirements for enzymatic formation of threonylcarbamoyladenosine (t6A) in tRNA: an in vivo study with Xenopus laevis oocytes.

We have investigated the specificity of the eukaryotic enzymatic machinery that transforms adenosine at position 37 (3' adjacent to anticodon) of several tRNAs into threonylcarbamoyladenosine (t6A37). To this end, 28 variants of yeast initiator tRNAMet and yeast tRNAVal, devoid of modified nucleotide, were produced by in vitro transcription with T7 polymerase of the corresponding synthetic tRNA genes and microinjected into the cytoplasm of Xenopus laevis oocytes. Threonylcarbamoyl incorporation was analyzed in tRNA transcripts mutated in the anticodon loop by substitution, deletion, or Insertion of nucleotides, or in the overall 3D structure of the tRNA by altering critical tertiary interactions. Specifically, we tested the effects of altering ribonucleotides in the anticodon loop, changes of the loop size, perturbations of the overall tRNA 3D structure due to mutations disruptive of the tertiary base pairs, and truncated tRNAs. The results indicate that, in addition to the targeted A37, only U36 was absolutely required. However, A38 in the anticodon loop considerably facilitates the quantitative conversion of A37 into t6A37 catalyzed by the enzymes present in X. laevis. The anticodon positions 34 and 35 were absolutely "neutral" and can accept any of the four canonical nucleotides A, U, C, or G. The anticodon loop size may vary from six to eight nucleotides, and the anticodon stem may have one mismatch pair of the type AxC or GxU at location 30-40 without affecting the efficiency of t6A37 formation and still t6A37 is efficiently formed. Although threonylcarbamoylation of A37 occurred with tRNA having limited perturbations of 3D structure, the overall L-shaped architecture of the tRNA substrate was required for efficient enzymatic conversion of A37 to t6A37. These results favor the idea that unique enzymatic machinery located in the oocyte cytoplasm catalyzes the formation of t6A37 in all U36A37-containing tRNAs (anticodon NNU). Microinjection of the yeast tRNAMeti into the cytoplasm of X. laevis oocytes also revealed the enzymatic activities for several other nucleotide modifications, respectively m1Gg, m2G10, m(2)2G26, m7G46, D47, m5C48/49, and m1A58