tRNA 3' processing endoribonuclease (3' tRNase) is an enzyme responsible for the removal of a 3' trailer from precursor tRNA (pre-tRNA). We purified approximately 85 kDa 3' tRNase from pig liver and determined its partial sequences. BLAST search of them suggested that the enzyme was the product of a candidate human prostate cancer susceptibility gene, ELAC2, the biological function of which was totally unknown. We cloned a human ELAC2 cDNA and expressed the ELAC2 protein in Escherichia coli. The recombinant ELAC2 was able to cleave human pre-tRNA(Arg) efficiently. The 3' tRNase activity of the yeast ortholog YKR079C was also observed. The C-terminal half of human ELAC2 was able to remove a 3' trailer from pre-tRNA(Arg), while the N-terminal half failed to do so. In the human genome exists a gene, ELAC1, which seems to correspond to the C-terminal half of 3' tRNase from ELAC2. We showed that human ELAC1 also has 3'-tRNase activity. Furthermore, we examined eight ELAC2 variants that seem to be associated with the occurrence of prostate cancer for 3'-tRNase activity. Seven ELAC2 variants which contain one to three amino acid substitutions showed efficient 3'-tRNase activities, while one truncated variant, which lacked a C-terminal half region, had no activity.

The tRNA 3-terminal CCA sequence is essential for aminoacylation of the tRNAs and for translation on the ribosome. The tRNAs are transcribed as larger precursor molecules containing 5 and 3 extra sequences. In the tRNAs that do not have the encoded CCA, the 3 extra sequence after the discriminator nucleotide is usually cleaved off by the tRNA 3 processing endoribonuclease (3 tRNase, or RNase Z), and the 3-terminal CCA residues are added thereto. Here we analyzed Thermotoga maritima 3 tRNase for enzymatic properties using various pre-tRNAs from T. maritima, in which all 46 tRNA genes encode CCA with only one exception. We found that the enzyme has the unprecedented activity that cleaves CCA-containing pre-tRNAs precisely after the CCA sequence, not after the discriminator. The assays for pre-tRNA variants suggest that the CA residues at nucleotides 75 and 76 are required for the enzyme to cleave pre-tRNAs after A at nucleotide 76 and that the cleavage occurs after nucleotide 75 if the sequence is not CA. Intriguingly, the pre-tRNA Met that is the only T. maritima pre-tRNA without the encoded CCA was cleaved after the discriminator. The kinetics data imply the existence of a CCA binding domain in T. maritima 3 tRNase. We also identified two amino acid residues critical for the cleavage site selection and several residues essential for the catalysis. Analysis of cleavage sites by 3 tRNases from another eubacteria Escherichia coli and two archaea Thermoplasma acidophilum and Pyrobaculum aerophilum corroborates the importance of the two amino acid residues for the cleavage site selection.Every single tRNA molecule ends with the sequence CCA (1). This 3Ј-terminal sequence is essential for aminoacylation of the tRNAs (2) and for translation on the ribosome (3) in all organisms. The tRNAs are transcribed as larger precursor molecules, which subsequently undergo various processing steps such as removal of 5Ј and 3Ј extra sequences to generate mature tRNAs (4).Because generally eukaryotic tRNA genes do not encode the CCA sequence, the eukaryotic tRNAs are supplemented with the CCA residues by tRNA nucleotidyltransferase (4, 5). It is believed that the discriminator nucleotide that protrudes from the aminoacyl stem, to which CCA is added, is generated by removing the 3Ј extra sequence primarily with tRNA 3Ј processing endoribonuclease (3Ј tRNase, or RNase Z) 1 (6 -14) and possibly in some circumstances with some unidentified exoribonucleases (15, 16).In contrast, the CCA sequences of all Escherichia coli tRNAs are encoded in its genome, and the six exoribonucleases RNase BN, RNase II, polynucleotide phosphorylase, RNase PH, RNase D, and RNase T are involved in the removal of 3Ј trailers to generate the CCA termini (17, 18). RNase II and polynucleotide phosphorylase, however, prefer unstructured RNAs such as mRNAs as substrates, so that their roles in tRNA maturation would probably be limited. In the other eubacteria and archaea, percentages of the CCA-coding tRNA genes vary with species from 0 to 100% (Table I). From the a...

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.

Transfer RNA (tRNA) 3' processing endoribonuclease (tRNase Z) is an enzyme responsible for the removal of a 3' trailer from pre-tRNA. There exists two types of tRNase Z: one is a short form (tRNase ZS) that consists of 300-400 amino acids, and the other is a long form (tRNase ZL) that contains 800-900 amino acids. Here we investigated whether the short and long forms have different preferences for various RNA substrates. We examined three recombinant tRNase ZSs from human, Escherichia coli and Thermotoga maritima, two recombinant tRNase ZLs from human and Saccharomyces cerevisiae, one tRNase ZL from pig liver, and the N- and C-terminal half regions of human tRNase ZL for cleavage of human micro-pre-tRNA(Arg) and the RNase 65 activity. All tRNase ZLs cleaved the micro-pre-tRNA and showed the RNase 65 activity, while all tRNase ZSs and both half regions of human tRNase ZL failed to do so with the exception of the C-terminal half, which barely cleaved the micro-pre-tRNA. We also show that only the long forms of tRNase Z can specifically cleave a target RNA under the direction of a new type of small guide RNA, hook RNA. These results indicate that indeed tRNase ZL and tRNase ZS have different substrate specificities and that the differences are attributed to the N-terminal half-domain of tRNase ZL. Furthermore, the optimal concentrations of NaCl, MgCl2 and MnCl2 differed between tRNase ZSs and tRNase ZLs, and the K(m) values implied that tRNase ZLs interact with pre-tRNA substrates more strongly than tRNase ZSs.

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.