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.     

Exosite modules guide substrate recognition in the ZiPD/ElaC protein family

Escherichia coli ZiPD is the best characterized protein encoded by the elaC gene family and is a model for the 3'-pre-tRNA processing endoribonucleases (tRNase Z). A metal ligand-based sequence alignment of ZiPD with metallo-beta-lactamase domain proteins of known crystallographic structure identifies a ZiPD-specific sequence insertion of approximately 50 residues, which we will refer to as the ZiPD exosite. Functionally characterized ZiPD homologs from Bacillus subtilis, Methanococcus janaschii, and human share the presence of the ZiPD exosite, which is also present in the amino-terminal, but not in the carboxyl-terminal, domain of ElaC2 proteins. Another class of functionally characterized tRNase Z enzymes from Thermotoga maritima and Arabidopsis thaliana lack characteristic motifs in the exosite but possess a sequence segment with clustered basic amino acid residues. As an experimental attempt to investigate the function of the exosite we constructed a ZiPD variant that lacks this module (ZiPDDelta). ZiPDDelta has almost wild-type-like catalytic properties for hydrolysis of the small, chromogenic substrate bis(p-nitrophenyl) phosphate. Removal of the ZiPD exosite only affects k(cat), which is reduced by less than 40%, whereas both K' andthe Hill coefficient (measures of the substrate affinity and cooperativity, respectively) remain unchanged. Hence, the exosite is not required for the intrinsic phosphodiesterase activity of ZiPD. Removal of the exosite also does not affect the dimerization properties of ZiPD. In contrast to the wild-type enzyme, ZiPDDelta does not process pre-tRNA, and gel shift assays demonstrate that only the wild-type enzyme, but not ZiPDDelta, binds mature tRNA. These findings show that the exosite is essential for pre-tRNA recognition. In conclusion, we identify a ZiPD exosite that guides physiological substrate recognition in the ZiPD/ElaC protein family.     

A novel endonucleolytic mechanism to generate the CCA 3' termini of tRNA molecules in Thermotoga maritima

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.     

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 beta-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 approximately 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.     

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.     

Substrate recognition ability differs among various prokaryotic tRNase Zs

There exists a significant difference in pre-tRNA preference among prokaryotic tRNase Zs. This is an enigma, because pre-tRNAs should form the common L-shaped structure and tRNase Zs should form the common structure based on the alphabeta/betaalpha-fold. To address this issue, we examined six different eubacterial and archaeal tRNase Zs including two newly isolated tRNase Zs for cleavage of 18 different pre-tRNA substrates. Two Thermotoga maritima, one Thermus thermophilus, one Bacillus subtilis, one Thermoplasma acidophilum, and one Pyrobaculum aerophilum enzymes were tested. To our surprise, the newly isolated proteins T. maritima and T. thermophilus showed the weak tRNase Z activity, even though their primary amino acid sequences are, on the whole, quite different from those of the typical tRNase Zs. We confirmed that substrate recognition ability is quite different among those tRNase Zs. In addition, we found that the optimal conditions as a whole differ significantly among the enzymes. From these results, we provided several clues to solve the enigma by showing the potential importance of the 74th-76th nucleotide sequence of pre-tRNA, the flexible arm length of tRNase Z, the divalent metal ion species, and the histidine corresponding His222 in T. maritima tRNase Z.     

Ribonuclease P RNA: models of the 15/16 bulge from Escherichia coli and the P15 stem loop of Bacillus subtilis.

The Escherichia coli ribonuclease P RNA 15/16 internal bulge loop and the Bacillus subtilis P15 stem loop are important substrate binding sites for the CCA-3' terminus of pre-tRNA. Models of E. coli 15/16 bulge loop and the B. subtilis P15 stem loop have been constructed using MC-SYM, a constraint satisfaction program. The models use covariation analysis data for suggesting initial base pairings, chemical probing, and protection/modification results to determine particular pairing orientations, and mutational experimental analysis data for tRNA-RNase P RNA contacts. The structures from E. coli and B. subtilis, although different in secondary structure, have similar sequence and function. Using MC-SYM, we are able to illustrate how the 3' end of the pre-tRNA is able to interact with this segment of the catalytic RNase P RNA. In addition, we propose additional hydrogen bonding between A76 in the 3' terminus of the tRNA and the 15/16 region of E. coli and to the loop of B. subtilis.     

Ribonuclease PH plays a major role in the exonucleolytic maturation of CCA-containing tRNA precursors in Bacillus subtilis

In contrast to Escherichia coli, where all tRNAs have the CCA motif encoded by their genes, two classes of tRNA precursors exist in the Gram-positive bacterium Bacillus subtilis. Previous evidence had shown that ribonuclease Z (RNase Z) was responsible for the endonucleolytic maturation of the 3' end of those tRNAs lacking an encoded CCA motif, accounting for about one-third of its tRNAs. This suggested that a second pathway of tRNA maturation must exist for those precursors with an encoded CCA motif. In this paper, we examine the potential role of the four known exoribonucleases of B.subtilis, PNPase, RNase R, RNase PH and YhaM, in this alternative pathway. In the absence of RNase PH, precursors of CCA-containing tRNAs accumulate that are a few nucleotides longer than the mature tRNA species observed in wild-type strains or in the other single exonuclease mutants. Thus, RNase PH plays an important role in removing the last few nucleotides of the tRNA precursor in vivo. The presence of three or four exonuclease mutations in a single strain results in CCA-containing tRNA precursors of increasing size, suggesting that, as in E.coli, the exonucleolytic pathway consists of multiple redundant enzymes. Assays of purified RNase PH using in vitro-synthesized tRNA precursor substrates suggest that RNase PH is sensitive to the presence of a CCA motif. The division of labor between the endonucleolytic and exonucleolytic pathways observed in vivo can be explained by the inhibition of RNase Z by the CCA motif in CCA-containing tRNA precursors and by the inhibition of exonucleases by stable secondary structure in the 3' extensions of the majority of CCA-less tRNAs.     

Endonucleolytic processing of CCA-less tRNA precursors by RNase Z in Bacillus subtilis

In contrast to Escherichia coli, where the 3' ends of tRNAs are primarily generated by exoribonucleases, maturation of the 3' end of tRNAs is catalysed by an endoribonuclease, known as RNase Z (or 3' tRNase), in many eukaryotic and archaeal systems. RNase Z cleaves tRNA precursors 3' to the discriminator base. Here we show that this activity, previously unsuspected in bacteria, is encoded by the yqjK gene of Bacillus subtilis. Decreased yqjK expression leads to an accumulation of a population of B.subtilis tRNAs in vivo, none of which have a CCA motif encoded in their genes, and YqjK cleaves tRNA precursors with the same specificity as plant RNase Z in vitro. We have thus renamed the gene rnz. A CCA motif downstream of the discriminator base inhibits RNase Z activity in vitro, with most of the inhibition due to the first C residue. Lastly, tRNAs with long 5' extensions are poor substrates for cleavage, suggesting that for some tRNAs, processing of the 5' end by RNase P may have to precede RNase Z cleavage.     

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.     

Naturally occurring mutations in human mitochondrial pre-tRNASer(UCN) can affect the transfer ribonuclease Z cleavage site, processing kinetics, and substrate secondary structure

tRNAs are transcribed as precursors with a 5' end leader and a 3' end trailer. The 5' end leader is processed by RNase P, and in most organisms in all three kingdoms, transfer ribonuclease (tRNase) Z can endonucleolytically remove the 3' end trailer. Long ((L)) and short ((S)) forms of the tRNase Z gene are present in the human genome. tRNase Z(L) processes a nuclear-encoded pre-tRNA approximately 1600-fold more efficiently than tRNase Z(S) and is predicted to have a strong mitochondrial transport signal. tRNase Z(L) could, thus, process both nuclear- and mitochondrially encoded pre-tRNAs. More than 150 pathogenesis-associated mutations have been found in the mitochondrial genome, most of them in the 22 mitochondrially encoded tRNAs. All the mutations investigated in human mitochondrial tRNA(Ser(UCN)) affect processing efficiency, and some affect the cleavage site and secondary structure. These changes could affect tRNase Z processing of mutant pre-tRNAs, perhaps contributing to mitochondrial disease.     

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).     

tRNase Z核酸内切酶的研究进展

tRNase Z是一种核酸内切酶,许多细菌、大多数真核生物以及所有的古细菌的tRNA3’末端加工过程都是由核酸内切酶tRNase Z催化的。tRNase Z能催化缺乏CCA的tRNA前体生成末尾带有核苷酸识别的3’-OH和5’磷酸尾巴的成熟tRNA。这对于CCA序列的添加、tRNA的氨酰化和蛋白质的合成十分重要。tRNase Z属于metallo-β-lactamases(MBL)超家族,存在短(tRNase ZS)和长(tRNase ZL)两种形式,具有tRNA 3’末端加工、引导定位蛋白、加工rRNA、与Rex2P的相互作用、调节细胞分化与分裂等功能。预期对tRNaseZ的功能和属性不断深入研究将会对AIDS和前列腺癌的治疗具有潜在和实际的推动作用。     

tRNA核酸内切酶的研究进展

tRNA在蛋白质合成过程中起着极其重要的作用。在所有的生物体内,tRNA首先以前体形式转录,然后必需经过一系列的加工后才能成为有功能的tRNA分子。tRNaseZ、RNaseP和tRNA剪接内切酶是参与tRNA前体加工的三种主要的核酸内切酶,分别参与tRNA前体3′末端、tRNA前体5′末端和内含子剪接的加工。这三种酶具有不同的结构特征,并且利用完全不同的催化机制水解磷酸二酯键。tRNaseZ和RNaseP都是金属酶,活性中心分别需要Zn^2＋和Mg^2＋的参与;而tRNA剪接内切酶活性中心不需要金属离子,是一个由不同催化亚基上的关键氨基酸残基构成的组合式活性中心。     

The RNase Z homologue encoded by Escherichia coli elaC gene is RNase BN

In eukaryotes, archaea, and in some eubacteria, removal of 3' precursor sequences during maturation of tRNA is catalyzed by an endoribonuclease, termed RNase Z. In contrast, in Escherichia coli, a variety of exoribonucleases carry out final 3' maturation. Yet, E. coli retains an RNase Z homologue, ElaC, whose function is under active study. We have overexpressed and purified to homogeneity His-tagged ElaC and show here that it is, in fact, the previously described enzyme, RNase BN. Thus, purified ElaC displays structural and catalytic properties identical to those ascribed to RNase BN. In addition, an elaC mutant strain behaves identically to a known RNase BN- strain, CAN. Finally, we show that wild type elaC can complement the mutation in strain CAN and that the elaC gene in strain CAN carries a nonsense mutation that results in loss of RNase BN activity. These data correct a previous misassignment for the gene encoding RNase BN. Based on the fact that the original RNase BN mutation has now been identified, we propose that the elaC gene be renamed rbn.     

Phylogenetic comparative chemical footprint analysis of the interaction between ribonuclease P RNA and tRNA.

Ribonuclease P RNA is the catalytic moiety of the ribonucleoprotein enzyme that endonucleolytically cleaves precursor sequences from the 5' ends of pre-tRNAs. The bacterial RNase P RNA-tRNA complex was examined with a footprinting approach, utilizing chemical modification to determine RNase P RNA nucleotides that potentially contact tRNA. RNase P RNA was modified with dimethylsulfate or kethoxal in the presence or absence of tRNA, and sites of modification were detected by primer extension. Comparison of the results reveals RNase P bases that are protected from modification upon binding tRNA. Analyses were carried out with RNase P RNAs from three different bacteria: Escherichia coli, Chromatium vinosum and Bacillus subtilis. Discrete bases of these RNAs that lie within conserved, homologous portions of the secondary structures are similarly protected. One protection among all three RNAs was attributed to the precursor segment of pre-tRNA. Experiments using pre-tRNAs containing precursor segments of variable length demonstrate that a precursor segment of only 2-4 nucleotides is sufficient to confer this protection. Deletion of the 3'-terminal CCA sequence of tRNA correlates with loss of protection of a particular loop in the RNase P RNA secondary structure. Analysis of mutant tRNAs containing sequential 3'-terminal deletions suggests a relative orientation of the bound tRNA CCA to that loop.     

Rp-phosphorothioate modifications in RNase P RNA that interfere with tRNA binding.

We have used Rp-phosphorothioate modifications and a binding interference assay to analyse the role of phosphate oxygens in tRNA recognition by Escherichia coli ribonuclease P (RNase P) RNA. Total (100%) Rp-phosphorothioate modification at A, C or G positions of RNase P RNA strongly impaired tRNA binding and pre-tRNA processing, while effects were less pronounced at U positions. Partially modified E. coli RNase P RNAs were separated into tRNA binding and non-binding fractions by gel retardation. Rp-phosphorothioate modifications that interfered with tRNA binding were found 5' of nucleotides A67, G68, U69, C70, C71, G72, A130, A132, A248, A249, G300, A317, A330, A352, C353 and C354. Manganese rescue at positions U69, C70, A130 and A132 identified, for the first time, sites of direct metal ion coordination in RNase P RNA. Most sites of interference are at strongly conserved nucleotides and nine reside within a long-range base-pairing interaction present in all known RNase P RNAs. In contrast to RNase P RNA, 100% Rp-phosphorothioate substitutions in tRNA showed only moderate effects on binding to RNase P RNAs from E. coli, Bacillus subtilis and Chromatium vinosum, suggesting that pro-Rp phosphate oxygens of mature tRNA contribute relatively little to the formation of the tRNA-RNase P RNA complex.