tRNase Z

Endonuclease tRNase Z catalyzes the generation of the mature 3' end of tRNA precursors through specific endonucleolytic cleavage. The enzyme has been characterized from organisms representative of all domains of life as well as from organelles, and the crystal structure of three bacterial enzymes has been determined. This review presents an overview of its properties and what is known about its structure, substrate recognition, cleavage site definition, and potential practical applications.     

Two archaeal tRNase Z enzymes: similar but different

The endoribonuclease tRNase Z plays an essential role in tRNA metabolism by removal of the 3' trailer element of precursor RNAs. To investigate tRNA processing in archaea, we identified and expressed the tRNase Z from Haloferax volcanii, a halophilic archaeon. The recombinant enzyme is a homodimer and efficiently processes precursor tRNAs. Although the protein is active in vivo at 2-4 M KCl, it is inhibited by high KCl concentrations in vitro, whereas 2-3 M (NH(4))(2)SO(4) do not inhibit tRNA processing. Analysis of the metal content of the metal depleted tRNase Z revealed that it still contains 0.4 Zn(2+) ions per dimer. In addition tRNase Z requires Mn(2+) ions for processing activity. We compared the halophilic tRNase Z to the homologous one from Pyrococcus furiosus, a thermophilic archaeon. Although both enzymes have 46% sequence similarity, they differ in their optimal reaction conditions. Both archaeal tRNase Z proteins process mitochondrial pre-tRNAs. Only the thermophilic tRNase Z shows in addition activity toward intron containing pre-tRNAs, 5' extended precursors, the phosphodiester bis(p-nitrophenyl)phosphate (bpNPP) and the glyoxalase II substrate S-D: -lactoylglutathion (SLG).     

Metal requirements and phosphodiesterase activity of tRNase Z enzymes

The endonuclease tRNase Z from A. thaliana (AthTRZ1) was originally isolated for its tRNA 3' processing activity. Here we show that AthTRZ1 also hydrolyzes the phosphodiester bond in bis(p-nitrophenyl) phosphate (bpNPP) with a kcat of 7.4 s-1 and a KM of 8.5 mM. We analyzed 22 variants of AthTRZ1 with respect to their ability to hydrolyze bpNPP. This mutational mapping identified fourteen variants that lost the ability to hydrolyze bpNPP and seven variants with reduced activity. Surprisingly, a single amino acid change (R252G) resulted in a ten times higher activity compared to the wild type enzyme. tRNase Z enzymes exist in long and short forms. We show here that in contrast to the short tRNase Z enzyme AthTRZ1, the long tRNase Z enzymes do not have bpNPP hydrolysis activity pointing to fundamental differences in substrate cleavage between the two enzyme forms. Furthermore, we determined the metal content of AthTRZ1 and analyzed the metal requirement for bpNPP hydrolysis. AthTRZ1 shows a high affinity for Zn2+ ions; even upon incubation with metal chelators, 0.76 Zn2+ ions are retained per dimer. In contrast to bpNPP hydrolysis, pre-tRNA processing requires additional metal ions, Mn2+ or Mg2+, as Zn2+ ions alone are insufficient.     

Unstructured RNA is a substrate for tRNase Z

tRNase Z, which exists in almost all cells, is believed to be working primarily for tRNA 3' maturation. In Escherichia coli, however, the tRNase Z gene appears to be dispensable under normal growth conditions, and its physiological role is not clear. Here, to investigate a possibility that E. coli tRNase Z cleaves RNAs other than pre-tRNAs, we tested several unstructured RNAs for cleavage. Surprisingly, all these substrates were cleaved very efficiently at multiple sites by a recombinant E. coli enzyme in vitro. tRNase Zs from Bacillus subtilis and Thermotoga maritima also cleaved various unstructured RNAs. The E. coli and B. subtilis enzymes seem to have a tendency to cleave after cytidine or before uridine, while cleavage by the T. maritima enzyme inevitably occurred after CCA in addition to the other cleavages. Assays to determine optimal conditions indicated that metal ion requirements differ between B. subtilis and T. maritima tRNase Zs. There was no significant difference in the observed rate constant between unstructured RNA and pre-tRNA substrates, while the K(d) value of a tRNase Z/unstructured RNA complex was much higher than that of an enzyme/pre-tRNA complex. Furthermore, eukaryotic tRNase Zs from yeast, pig, and human cleaved unstructured RNA at multiple sites, but an archaeal tRNase Z from Pyrobaculum aerophilum did not.     

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 Z^S) is the only one found in bacteria and archaebacteria and is also present in some eukaryotes. The homologous long form (tRNase Z^L), exclusively found in eukaryotes, consists of related amino- and carboxy-domains, suggesting that tRNase Z^L arose from a tandem duplication of tRNase Z^S followed by interdependent divergence of the domains. X-ray crystallographic structures of tRNase Z^S 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 Z^L structures have been solved; alternative biophysical studies are therefore needed to illuminate its functional characteristics. Structural analyses of tRNase Z^L 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 Z^L function.     

Human cytosolic tRNase Z can downregulate gene expression through miRNA

A long form of tRNase Z (tRNase Z^L) can cleave any target RNA at any desired site under the direction of artificial small guide RNA including ∼25-nucleotide hook-shaped RNA. Here we show that human miR-103 can form a hook structure to guide target RNA cleavage by human cytosolic tRNase Z^L in vitro. In vivo analyses using luciferase mRNAs modified to contain miR-103 target sequences in their 3′ untranslated regions indicated that miR-103 downregulates gene expression through directing mRNA cleavage by tRNase Z^L. The present data suggest the possibility that human cytosolic tRNase Z^L modulates gene expression through a subset of microRNAs in the cells.     

Identification and Sequence Analysis of Metazoan tRNA 3'-End Processing Enzymes tRNase Zs

tRNase Z is the endonuclease responsible for removing the 3'-trailer sequences from precursor tRNAs, a prerequisite for the addition of the CCA sequence. It occurs in the short (tRNase Z(S)) and long (tRNase Z(L)) forms. Here we report the identification and sequence analysis of candidate tRNase Zs from 81 metazoan species. We found that the vast majority of deuterostomes, lophotrochozoans and lower metazoans have one tRNase Z(S) and one tRNase Z(L) genes, whereas ecdysozoans possess only a single tRNase Z(L) gene. Sequence analysis revealed that in metazoans, a single nuclear tRNase Z(L) gene is likely to encode both the nuclear and mitochondrial forms of tRNA 3'-end processing enzyme through mechanisms that include alternative translation initiation from two in-frame start codons and alternative splicing. Sequence conservation analysis revealed a variant PxKxRN motif, PxPxRG, which is located in the N-terminal region of tRNase Z(S)s. We also identified a previously unappreciated motif, AxDx, present in the C-terminal region of both tRNase Z(S)s and tRNase Z(L)s. The AxDx motif consisting mainly of a very short loop is potentially close enough to form hydrogen bonds with the loop containing the PxKxRN or PxPxRG motif. Through complementation analysis, we demonstrated the likely functional importance of the AxDx motif. In conclusion, our analysis supports the notion that in metazoans a single tRNase Z(L) has evolved to participate in both nuclear and mitochondrial tRNA 3'-end processing, whereas tRNase Z(S) may have evolved new functions. Our analysis also unveils new evolutionarily conserved motifs in tRNase Zs, including the C-terminal AxDx motif, which may have functional significance.     

tRNase Z catalysis and conserved residues on the carboxy side of the His cluster

tRNAs are transcribed as precursors and processed in a series of required reactions leading to aminoacylation and translation. The 3'-end trailer can be removed by the pre-tRNA processing endonuclease tRNase Z, an ancient, conserved member of the beta-lactamase superfamily of metal-dependent hydrolases. The signature sequence of this family, the His domain (HxHxDH, Motif II), and histidines in Motifs III and V and aspartate in Motif IV contribute seven side chains for the coordination of two divalent metal ions. We previously investigated the effects on catalysis of substitutions in Motif II and in the PxKxRN loop and Motif I on the amino side of Motif II. Herein, we present the effects of substitutions on the carboxy side of Motif II within Motifs III, IV, the HEAT and HST loops, and Motif V. Substitution of the Motif IV aspartate reduces catalytic efficiency more than 10,000-fold. Histidines in Motif III, V, and the HST loop are also functionally important. Strikingly, replacement of Glu in the HEAT loop with Ala reduces efficiency by approximately 1000-fold. Proximity and orientation of this Glu side chain relative to His in the HST loop and the importance of both residues for catalysis suggest that they function as a duo in proton transfer at the final stage of reaction, characteristic of the tRNase Z class of RNA endonucleases.     

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-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.     

The tRNase Z family of proteins: physiological functions, substrate specificity and structural properties

tRNase Z is the endoribonuclease that generates the mature 3'-end of tRNA molecules by removal of the 3'-trailer elements of precursor tRNAs. This enzyme has been characterized from representatives of all three domains of life (Bacteria, Archaea and Eukarya), as well as from mitochondria and chloroplasts. tRNase Z enzymes come in two forms: short versions (280-360 amino acids in length), present in all three kingdoms, and long versions (750-930 amino acids), present only in eukaryotes. The recently solved crystal structure of the bacterial tRNase Z provides the structural basis for the understanding of central functional elements. The substrate is recognized by an exosite that protrudes from the main protein body and consists of a metallo-beta-lactamase domain. Cleavage of the precursor tRNA occurs at the binuclear zinc site located in the other subunit of the functional homodimer. The first gene of the tRNase Z family was cloned in 2002. Since then a comprehensive set of data has been acquired concerning this new enzyme, including detailed functional studies on purified recombinant enzymes, mutagenesis studies and finally the determination of the crystal structure of three bacterial enzymes. This review summarizes the current knowledge about these exciting enzymes.     

An Auxin-Regulated Gene of Arabidopsis thaliana Encodes a DNA-Binding Protein

We have isolated a single-copy gene from the plant Arabidopsis thaliana, called dbp, which encodes a lysine-rich, DNA-binding protein. The Dbp protein has a molecular weight and a composition resembling histone H1. When the dbp gene was expressed in bacteria, the protein product bound DNA nonspecifically. The dbp gene is expressed constitutively in all parts of the plant but is induced five times above this basal level in apical zones. In vitro hormone-depletion experiments showed that the expression in the shoot apex could be induced by exogenous auxin. In situ hybridizations in the root apex indicated that the expression of dbp is enhanced in the region of cell division.     

The tRNA 30-end Processing Enzyme tRNase Z2 Contributes to Chloroplast Biogenesis in Rice

tRNase Z (TRZ) is a ubiquitous endonuclease that removes the 30-trailer from precursor tRNAs during maturation. In yeast and animals, TRZ regulates the cell cycle via its (t)RNA processing activity;how...     

Analysis of the functional modules of the tRNA 3' endonuclease (tRNase Z)

tRNA 3' processing is one of the essential steps during tRNA maturation. The tRNA 3'-processing endonuclease tRNase Z was only recently isolated, and its functional domains have not been identified so far. We performed an extensive mutational study to identify amino acids and regions involved in dimerization, tRNA binding, and catalytic activity. 29 deletion and point variants of the tRNase Z enzyme were generated. According to the results obtained, variants can be sorted into five different classes. The first class still had wild type activity in all three respects. Members of the second and third class still formed dimers and bound tRNAs but had reduced catalytic activity (class two) or no catalytic activity (class three). The fourth class still formed dimers but did not bind the tRNA and did not process precursors. Since this class still formed dimers, it seems that the amino acids mutated in these variants are important for RNA binding. The fifth class did not have any activity anymore. Several conserved amino acids could be mutated without or with little loss of activity.     

Phytoalexin-Deficient Mutants of Arabidopsis Reveal That Pad4 Encodes a Regulatory Factor and That Four Pad Genes Contribute to Downy Mildew Resistance

We are working to determine the role of the Arabidopsis phytoalexin, camalexin, in protecting the plant from pathogen attack by isolating phytoalexin-deficient (pad) mutants in the accession Columbia (Col-0) and examining their response to pathogens. Mutations in PAD1, PAD2, and PAD4 caused enhanced susceptibility to the bacterial pathogen Pseudomonas syringae pv. maculicola strain ES4326 (PsmES4326), while mutations in PAD3 or PAD5 did not. Camalexin was not detected in any of the double mutants pad1-1 pad2-1, pad1-1 pad3-1 or pad2-1 pad3-1. Growth of PsmES4326 in pad1-1 pad2-1 was greater than that in pad1-1 or pad2-1 plants, while growth in pad1-1 pad3-1 and pad2-1 pad3-1 plants was similar to that in pad1-1 and pad2-1 plants, respectively. The pad4-1 mutation caused reduced camalexin synthesis in response to PsmES4326 infection, but not in response to Cochliobolus carbonum infection, indicating that PAD4 has a regulatory function. PAD1, PAD2, PAD3 and PAD4 are all required for resistance to the eukaryotic biotroph Peronospora parasitica. The pad4-1 mutation caused the most dramatic change, exhibiting full susceptibility to four of six Col-incompatible parasite isolates. Interestingly, each combination of double mutants between pad1-1, pad2-1 and pad3-1 exhibited additive shifts to moderate or full susceptibility to most of the isolates.     

The N-terminal half-domain of the long form of tRNase Z is required for the RNase 65 activity

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.     

The flexible arm of tRNase Z is not essential for pre-tRNA binding but affects cleavage site selection

tRNase Z is an enzyme responsible for removing a 3′ trailer from pre-tRNA. Although most tRNase Zs cleave pre-tRNAs immediately after the discriminator nucleotide with the exception of Thermotoga maritima tRNase Z, which cleaves after the ₇₄CCA₇₆ sequence, our knowledge was limited about how the cleavage site in pre-tRNA is selected. Bacterial tRNase Zs contain a unique domain termed flexible arm, which extends from the core domain. Using various tRNase Z variants, here we examined how the flexible arm affects the cleavage site selection. T. maritima tRNase Z variants with modified flexible arms shifted the cleavage site and a Bacillus subtilis tRNase Z variant with no flexible arm showed an anomalous cleavage activity. Some of the T. maritima/B. subtilis chimeric enzymes had both properties: they recognized ₇₄CCA₇₆-containing pre-tRNA and cleaved it after the discriminator. Taken together, the present data indicate that the flexible arm is not essential for pre-tRNA binding but affects the cleavage site selection probably by pushing the distal region of the T arm in such a way that the discriminator nucleotide becomes closer to the catalytic site.     

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.     

紫外线强烈诱导的谷胱甘肽转移酶基因的功能鉴定

植物谷胱甘肽转移酶（glutathione S-transferases,GSTs)基因家族在逆境反应和植物生长发育过程中都起着非常重要的作用。为了阐明GST在紫外辐射下是否对植物有保护作用,以紫外强烈诱导表达的GST、cDNA为探针,筛选拟南芥cDNA文库,获得了这种GST的全长cDNA;利用此cDNA构建植物表达载体,并通过农杆菌介导法转化拟南芬,使其在拟南芥中得到大量表达;通过对转基因植株的紫外辐射耐性分析,证实了该GST的过量表达可明显增强拟南芥对紫外辐射损伤作用的耐受性。