Asako Minagawa

Crystal Structure of the tRNA 3′ Processing Endoribonuclease tRNase Z from Thermotoga maritima

The maturation of the tRNA 3′ end is catalyzed by a tRNA 3′ processing endoribonuclease named tRNase Z (RNase Z or 3′-tRNase) in eukaryotes, Archaea, and some bacteria. The tRNase Z generally cuts the 3′ extra sequence from the precursor tRNA after the discriminator nucleotide. In contrast, Thermotoga maritima tRNase Z cleaves the precursor tRNA precisely after the CCA sequence. In this study, we determined the crystal structure of T. maritima tRNase Z at 2.6-Å resolution. The tRNase Z has a four-layer αβ/βα sandwich fold, which is classified as a metallo-β-lactamase fold, and forms a dimer. The active site is located at one edge of the β-sandwich and is composed of conserved motifs. Based on the structure, we constructed a docking model with the tRNAs that suggests how tRNase Z may recognize the substrate tRNAs.

Identification by Mn2+ rescue of two residues essential for the proton transfer of tRNase Z catalysis

Thermotoga maritima tRNase Z cleaves pre-tRNAs containing the 74CCA76 sequence precisely after the A76 residue to create the mature 3′ termini. Its crystal structure has revealed a four-layer αβ/βα sandwich fold that is typically found in the metallo-β-lactamase superfamily. The well-conserved six histidine and two aspartate residues together with metal ions are assumed to form the tRNase Z catalytic center. Here, we examined tRNase Z variants containing single amino acid substitutions in the catalytic center for pre-tRNA cleavage. Cleavage by each variant in the presence of Mg2+ was hardly detected, although it is bound to pre-tRNA. Surprisingly, however, Mn2+ ions restored the lost Mg2+-dependent activity with two exceptions of the Asp52Ala and His222Ala substitutions, which abolished the activity almost completely. These results provide a piece of evidence that Asp-52 and His-222 directly contribute the proton transfer for the catalysis.