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

Ryohei Ishii,Asako Minagawa,Hiroaki Takaku,Masamichi Takagi,Masayuki Nashimoto,Shigeyuki Yokoyama

doi:10.1074/jbc.m500355200

Abstract

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-A resolution. The tRNase Z has a four-layer alphabeta/betaalpha sandwich fold, which is classified as a metallo-beta-lactamase fold, and forms a dimer. The active site is located at one edge of the beta-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.

Highlights

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 eukaryotic 3Ј processing is done mainly by a tRNA 3Ј processing endoribonuclease named tRNase Z (EC 3.1.26.11; RNase Z or 3Ј-tRNase), which cuts the pre-tRNAs with the 3Ј extra sequence after the
Protein Expression and Purification—The double-stranded DNA encoding the T. maritima tRNase Z gene was amplified by PCR from the plasmid pQE/Tm(WT), which produces a histidine-tagged tRNase Z protein [15], to remove the tag-encoding sequence and was cloned between the NdeI and SalI sites of the expression vector pET26b(ϩ) (Novagen)

Summary

Unique reflections

Completeness, all data (last shell) (%) I/␴(I), all data (last shell) Rsym,a all data (last shell) (%) Refinement. A Rsym ϭ ⌺͉Ihkl ϪIhkl͘/⌺Ihkl whereIhklis the mean value of Ihkl. B The free R factor was calculated using 5% of reflections omitted from the refinement A Rsym ϭ ⌺͉Ihkl ϪIhkl͘/⌺Ihkl whereIhklis the mean value of Ihkl. b The free R factor was calculated using 5% of reflections omitted from the refinement

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

These five conserved motifs are also conserved in the tRNase