Tethered Domains and Flexible Regions in tRNase ZL, the Long Form of tRNase Z

tRNase Z, a member of the metallo-β-lactamase family, endonucleolytically removes the pre-tRNA 3′ trailer in a step central to tRNA maturation. The short form (tRNase ZS) is the only one found in bacteria and archaebacteria and is also present in some eukaryotes. The homologous long form (tRNase ZL), exclusively found in eukaryotes, consists of related amino- and carboxy-domains, suggesting that tRNase ZL arose from a tandem duplication of tRNase ZS followed by interdependent divergence of the domains. X-ray crystallographic structures of tRNase ZS reveal a flexible arm (FA) extruded from the body of tRNase Z remote from the active site that binds tRNA far from the scissile bond. No tRNase ZL structures have been solved; alternative biophysical studies are therefore needed to illuminate its functional characteristics. Structural analyses of tRNase ZL performed by limited proteolysis, two dimensional gel electrophoresis and mass spectrometry establish stability of the amino and carboxy domains and flexibility of the FA and inter-domain tether, with implications for tRNase ZL function

Substitutions in conserved regions preceding and within the linker affect activity and flexibility of tRNase ZL, the long form of tRNase Z

The enzyme tRNase Z, a member of the metallo-β-lactamase family, endonucleolytically removes 3\u27 trailers from precursor tRNAs, preparing them for CCA addition and aminoacylation. The short form of tRNase Z, tRNase ZS, functions as a homodimer and is found in all prokaryotes and some eukaryotes. The long form, tRNase ZL, related to tRNase ZS through tandem duplication and found only in eukaryotes, possesses ~2,000-fold greater catalytic efficiency than tRNase ZS. tRNase ZL consists of related but diverged amino and carboxy domains connected by a flexible linker (also referred to as a flexible tether) and functions as a monomer. The amino domain retains the flexible arm responsible for substrate recognition and binding while the carboxy domain retains the active site. The linker region was explored by Ala-scanning through two conserved regions of D. melanogaster tRNase Z: NdomTprox, located at the carboxy end of the amino domain proximal to the linker, and Tflex, a flexible site in the linker. Periodic substitutions in a hydrophobic patch (F329 and L332) at the carboxy end of NdomTprox show 2,700 and 670-fold impairment relative to wild type, respectively, accompanied by reduced linker flexibility at N-T inside the Ndom- linker boundary. The Ala substitution for N378 in the Tflex region has 10-fold higher catalytic efficiency than wild type and locally decreased flexibility, while the Ala substitution at R382 reduces catalytic efficiency ~50-fold. These changes in pre-tRNA processing kinetics and protein flexibility are interpreted in light of a recent crystal structure for S. cerevisiae tRNase Z, suggesting transmission of local changes in hydrophobicity into the skeleton of the amino domain

Mutations in ELAC2 associated with hypertrophic cardiomyopathy impair mitochondrial tRNA 3'-end processing.

Mutations in either the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial respiration. Within this group, an increasing number of mutations have been identified in nuclear genes involved in mitochondrial RNA metabolism, including ELAC2. The ELAC2 gene codes for the mitochondrial RNase Z, responsible for endonucleolytic cleavage of the 3' ends of mitochondrial pre-tRNAs. Here, we report the identification of 16 novel ELAC2 variants in individuals presenting with mitochondrial respiratory chain deficiency, hypertrophic cardiomyopathy (HCM), and lactic acidosis. We provide evidence for the pathogenicity of the novel missense variants by studying the RNase Z activity in an in vitro system. We also modeled the residues affected by a missense mutation in solved RNase Z structures, providing insight into enzyme structure and function. Finally, we show that primary fibroblasts from the affected individuals have elevated levels of unprocessed mitochondrial RNA precursors. Our study thus broadly confirms the correlation of ELAC2 variants with severe infantile-onset forms of HCM and mitochondrial respiratory chain dysfunction. One rare missense variant associated with the occurrence of prostate cancer (p.Arg781His) impairs the mitochondrial RNase Z activity of ELAC2, suggesting a functional link between tumorigenesis and mitochondrial RNA metabolism.Includes MRC, BBSRC, Wellcome Trust and NIHR

Residues in two homology blocks on the amino side of the tRNase Z His domain contribute unexpectedly to pre-tRNA 3′ end processing

tRNase Z, which can endonucleolytically remove pre-tRNA 3′-end trailers, possesses the signature His domain (HxHxDH; Motif II) of the β-lactamase family of metal-dependent hydrolases. Motif II combines with Motifs III–V on its carboxy side to coordinate two divalent metal ions, constituting the catalytic core. The PxKxRN loop and Motif I on the amino side of Motif II have been suggested to modulate tRNase Z activity, including the anti-determinant effect of CCA in mature tRNA. Ala walks through these two homology blocks reveal residues in which the substitutions unexpectedly reduce catalytic efficiency. While substitutions in Motif II can drastically affect k(cat) without affecting k(M), five- to 15-fold increases in k(M) are observed with substitutions in several conserved residues in the PxKxRN loop and Motif I. These increases in k(M) suggest a model for substrate binding. Expressed tRNase Z processes mature tRNA with CCA at the 3′ end ∼80 times less efficiently than a pre-tRNA possessing natural sequence of the 3′-end trailer, due to reduced k(cat) with no effect on k(M), showing the CCA anti-determinant to be a characteristic of this enzyme

Mitochondrial tRNA 3′ end metabolism and human disease

Over 150 mutations in the mitochondrial genome have been shown to be associated with human disease. Remarkably, two-thirds of them are found in tRNA genes, which constitute only one-tenth of the mitochondrial genome. A total of 22 tRNAs punctuate the genome and are produced together with 11 mRNAs and 2 rRNAs from long polycistronic primary transcripts with almost no spacers. Pre-tRNAs thus require precise endonucleolytic excision. Furthermore, the CCA triplet which forms the 3′ end of all tRNAs is not encoded, but must be synthesized by the CCA-adding enzyme after 3′ end cleavage. Amino acid attachment to the CCA of mature tRNA is performed by aminoacyl-tRNA synthetases, which, like the preceding processing enzymes, are nuclear-encoded and imported into mitochondria. Here, we critically review the effectiveness and reliability of evidence obtained from reactions with in vitro transcripts that pathogenesis-associated mutant mitochondrial tRNAs can lead to deficiencies in tRNA 3′ end metabolism (3′ end cleavage, CCA addition and aminoacylation) toward an understanding of molecular mechanisms underlying human tRNA disorders. These defects probably contribute, individually and cumulatively, to the progression of human mitochondrial diseases