Distinct tRNA modifications in the thermo-acidophilic archaeon, Thermoplasma acidophilum

Thermoplasma acidophilum is a thermo-acidophilic archaeon. We purified tRNA^Leu (UAG) from T. acidophilum using a solid-phase DNA probe method and determined the RNA sequence after determining via nucleoside analysis and m⁷G-specific aniline cleavage because it has been reported that T. acidophilum tRNA contains m⁷G, which is generally not found in archaeal tRNAs. RNA sequencing and liquid chromatography–mass spectrometry revealed that the m⁷G modification exists at a novel position 49. Furthermore, we found several distinct modifications, which have not previously been found in archaeal tRNA, such as 4-thiouridine9, archaeosine13 and 5-carbamoylmethyuridine34. The related tRNA modification enzymes and their genes are discussed.     

Effects of tRNALeu1 overproduction in Escherichia coli

Strains of Escherichia coli have been produced which express very high levels of the tRNA^leu₁ isoacceptor. This was accomplished by transforming cells with plasmids containing the leuV operon which encodes three copies of the tRNA^Leu₁ gene. Most transformants grew very slowly and exhibited a 15-fold increase in cellular concentrations of tRNA^Leu₁ As a result, total cellular tRNA concentration was approximately doubled and 56% of the total was tRNA^Leu₁. We examined a number of parameters which might be expected to be affected by imbalances in tRNA concentration: in vivo tRNA charging levels, misreading, ribosome step time, and tRNA modification. Surprisingly, no increase in intracellular ppGpp levels was detected even though only about 40% of total leucyl tRNA was found to be charged in vivo. Gross ribosomal misreading was not detected, and it was shown that ribosomal step times were reduced between two- and threefold. Analyses of leucyl tRNA isolated from these slow-growing strains showed that at least 90% of the detectable tRNA^Leu₁ was hypomodified as judged by altered mobility on RPC-5 reverse-phase columns, and by specific modification assays using tRNA(m¹G)-methyltransferase and pseudo-uridylate synthetase. Analysis of fast-growing revertants demonstrated that tRNA concentration per se may not explain growth inhibition because selected revertants which grew at wild-type growth rates displayed levels of tRNA comparable to that of control strains bearing the leuV operon. A synthetic tRNA^Leu₁ operon under the control of the T7 promoter was prepared which, when induced, produced six- to sevenfold increases in tRNA^Leu₁ levels. This level of tRNA^Leu₁ titrated the modification system as judged by RPC-5 column chromatography. Overall, our results suggest that hypomodified tRNA may explain, in part, the observed effects on growth, and that the protein-synthesizing system can tolerate an enormous increase in the concentration of a single tRNA.     

Overproduction and purification ofEscherichia coli tRNALeu

Chemically synthesized genes encodingEscherichia coli tRNA ₁ ^Leu and tRNA ₂ ^Leu were ligated into the plasmid pTrc99B. then transformed intoEscherichia coli MT102, respectively. The positive transformants, named MT-Leu1 and MT-Leu2, were confirmed by DNA sequencing, and the conditions of cultivation for the two transformants were optimized. As a result, leucinc accepting activity of their total tRNA reached 810 and 560 pmol/A₂₆₀, respectively: the content of tRNA ₁ ^Leu was 50% of total tRNA from MT-Leu1, while that of tRNA ₂ ^Leu was 30% of total tRNA from MT-Leu2. Both tRNA^Leus from their rotal tRNs were fractionated to 1 600 pmol/A₂₆₀ after DEAE-Sepharose and BD-cellulose column chromatography. The accurate kinetic constants of aminoacylation of the two isoacceptors of tRNA^Leu catalyzed by leucyl-tRNA synthetase were determined.     

Novel Temperature-Sensitive Mutants of Escherichia coli That Are Unable To Grow in the Absence of Wild-Type tRNA6Leu

Escherichia coli has only a single copy of a gene for tRNA₆^Leu (Y. Komine et al., J. Mol. Biol. 212:579–598, 1990). The anticodon of this tRNA is CAA (the wobble position C is modified to O²-methylcytidine), and it recognizes the codon UUG. Since UUG is also recognized by tRNA₄^Leu, which has UAA (the wobble position U is modified to 5-carboxymethylaminomethyl-O²-methyluridine) as its anticodon, tRNA₆^Leu is not essential for protein synthesis. The BT63 strain has a mutation in the anticodon of tRNA₆^Leu with a change from CAA to CUA, which results in the amber suppressor activity of this strain (supP, Su⁺6). We isolated 18 temperature-sensitive (ts) mutants of the BT63 strain whose temperature sensitivity was complemented by introduction of the wild-type gene for tRNA₆^Leu. These tRNA₆^Leu-requiring mutants were classified into two groups. The 10 group I mutants had a mutation in the miaA gene, whose product is involved in a modification of tRNAs that stabilizes codon-anticodon interactions. Overexpression of the gene for tRNA₄^Leu restored the growth of group I mutants at 42°C. Replacement of the CUG codon with UUG reduced the efficiency of translation in group I mutants. These results suggest that unmodified tRNA₄^Leu poorly recognizes the UUG codon at 42°C and that the wild-type tRNA₆^Leu is required for translation in order to maintain cell viability. The mutations in the six group II mutants were complemented by introduction of the gidA gene, which may be involved in cell division. The reduced efficiency of translation caused by replacement of the CUG codon with UUG was also observed in group II mutants. The mechanism of requirement for tRNA₆^Leu remains to be investigated.In the universal genetic code, 61 sense codons correspond to 20 amino acids, and the various tRNA species mediate the flow of information from the genetic code to amino acid sequences. Since codon-anticodon interactions permit wobble pairing at the third position, 32 tRNAs, including tRNA^fMet, should theoretically be sufficient for a complete translation system. Although some organisms have fewer tRNAs (1), most have abundant tRNA species and multiple copies of major tRNAs. For example, Escherichia coli has 86 genes for tRNA (79 genes identified in reference 14, 6 new ones reported in reference 3, and one fMet tRNA at positions 2945406 to 2945482) that encode 46 different amino acid acceptor species. Although abundant genes for tRNAs are probably required for efficient translation, the significance of the apparently nonessential tRNAs has not been examined.E. coli has five isoaccepting species of tRNA^Leu. According to the wobble rule, tRNA₁^Leu recognizes only the CUG codon. The CUG codon is also recognized by tRNA₃^Leu (tRNA₂^Leu) and thus tRNA₁^Leu may not be essential for protein synthesis. Similarly, tRNA₆^Leu is supposed to recognize only the UUG codon, but tRNA₄^Leu can recognize both UUA and UUG codons. Thus, tRNA₆^Leu appears to be dispensable. The existence of an amber suppressor mutation of tRNA₆^Leu (supP, Su⁺6) supports this possibility. tRNA₆^Leu is encoded by a single-copy gene, leuX (supP), and Su⁺6 has a mutation in the anticodon, which suggests loss of the ability to recognize UUG (26). Why are so many species of tRNA^Leu required? Holmes et al. (12) examined the utilization of the isoaccepting species of tRNA^Leu in protein synthesis and showed that utilization differs depending on the growth medium; in minimal medium, isoacceptors tRNA₂^Leu (cited as tRNA₃^Leu; see Materials and Methods) and tRNA₄^Leu are the predominant species that are found bound to ribosomes, but an increased relative level of tRNA₁^Leu is found bound to ribosomes in rich medium. The existence of tRNA₆^Leu is puzzling. This isoaccepting tRNA accounts for approximately 10% of the tRNA^Leu in total-cell extracts. However, little if any tRNA₆^Leu is found on ribosomes in vivo, and it is also only weakly active in protein synthesis in vitro with mRNA from E. coli (12). It thus appears that tRNA₆^Leu is only minimally involved in protein synthesis in E. coli.To investigate the role of tRNA₆^Leu in E. coli, we attempted to isolate tRNA₆^Leu-requiring mutants from an Su⁺6 strain. These mutants required wild-type tRNA₆^Leu for survival at a nonpermissive temperature. We report here the isolation and the characterization of these mutants.     

Tritium exchange studies of transfer RNA in native and denaturated conformations

Tritium exchange was used as a probe of transfer RNA structure in experiments with unfractionated tRNA (tRNA^Unfrac and homogeneous tRNA₃^Leu from bakers' yeast. Exchange kinetics were measured over a range of ionic conditions that vary in ability to stabilize the secondary and tertiary structure of tRNA. The native conformations of both samples show the same kinetics of exchange. The kinetics for tRNA₃^Leu trapped in a denatured state in a “native” solvent are much faster, reflecting the conformation and not the ionic medium. In 0.1 M-Na⁺, where tRNA₃^Leu is denatured, the kinetics for tRNA^Unfrac are intermediate between those for native and denatured tRNA₃^Leu, suggesting that in this solvent at 0 °C some tRNAs are denatured whereas other are still native. Upon further lowering of Na⁺ concentration, tRNA^Unfrac shows increasingly faster exchange, suggesting complete electrostatic denaturation of the tertiary structure of all the tRNAs in the sample, and even disruption of secondary structure.Extrapolation of the essentially linear early-time kinetics to zero time provides minimal estimates of the number of slowly exchanging hydrogens. For native tRNA₃^Leu the number is 111±2 hydrogens, whereas for the trapped denatured conformation it is only 95±2. This difference reflects a smaller number of hydrogen-bonded bases in the denatured conformation. In 1 M-Na⁺, 101±2 slowly exchanging hydrogens are found for the native tRNA₃^Leu conformation, suggesting an incompletely formed native structure. For native tRNA^Unfrac the comparable number is 101±3. These numbers of slowly exchanging hydrogens in the native conformations are consistent with tertiary structural hydrogen-bonding. Furthermore, this tertiary structure must be responsible for the slower exchange by native tRNA. The observed numbers of exchangeable hydrogens provide a basis for comparison of hydrogen-bonding interactions in native and denatured tRNA conformations.The mechanism of renaturation was also investigated, using tritium exchange as a monitor of perturbation of base pairing during the transition. When tRNA^Unfrac in low Na⁺ is renatured by addition of Mg²⁺ during tritium exchangeout, a burst of exchange or “spillage” of tritium is detected. This suggests that a fraction of the base pairs of the rapidly renaturing tRNAs in the mixture is disrupted during renaturation. In that event, and by analogy with tRNA₃^Leu, part of the base-pairing arrangement of the denatured conformations may not be preserved in the native state; and if the native conformation includes the full “cloverleaf” pattern of secondary structure, that pattern may not be intact in some denatured conformations.     

Biosynthesis of 7-deazaguanosine-modified tRNA nucleosides: a new role for GTP cyclohydrolase I

Queuosine (Q) and archaeosine (G⁺) are hypermodified ribonucleosides found in tRNA. Q is present in the anticodon region of tRNA_GUN in Eukarya and Bacteria, while G⁺ is found at position 15 in the D-loop of archaeal tRNA. Prokaryotes produce these 7-deazaguanosine derivatives de novo from GTP through the 7-cyano-7-deazaguanine (pre-Q₀) intermediate, but mammals import the free base, queuine, obtained from the diet or the intestinal flora. By combining the results of comparative genomic analysis with those of genetic studies, we show that the first enzyme of the folate pathway, GTP cyclohydrolase I (GCYH-I), encoded in Escherichia coli by folE, is also the first enzyme of pre-Q₀ biosynthesis in both prokaryotic kingdoms. Indeed, tRNA extracted from an E. coli ΔfolE strain is devoid of Q and the deficiency is complemented by expressing GCYH-I-encoding genes from different bacterial or archaeal origins. In a similar fashion, tRNA extracted from a Haloferax volcanii strain carrying a deletion of the GCYH-I-encoding gene contains only traces of G⁺. These results link the production of a tRNA-modified base to primary metabolism and further clarify the biosynthetic pathway for these complex modified nucleosides.     

Bacteriophage T4 tRNA<Superscript>Leu</Superscript>

WHEN T4 bacteriophage infects Escherichia coli, the host tRNA complement is altered in two ways: (1) a tRNA^Leu is inactivated by endonucleolytic cleavage^1–3 and sequencing has shown that this tRNA recognizes the codon CUG^4,5; (2) seven or eight new tRNA species are introduced by the T4 genome^6–8. One of these, a leucine tRNA, differs from all the host species of tRNA^Leu, having different chromatographic properties^9,10, being labelled preferentially with radioactive ³⁵SO₄ following phage infection^6,11 and specifically hybridizing to T4 DNA^7,8,11,12.     

Crystal structure of the archaeosine synthase QueF‐like—Insights into amidino transfer and tRNA recognition by the tunnel fold

The tunneling‐fold (T‐fold) structural superfamily has emerged as a versatile protein scaffold of diverse catalytic activities. This is especially evident in the pathways to the 7‐deazaguanosine modified nucleosides of tRNA queuosine and archaeosine. Four members of the T‐fold superfamily have been confirmed in these pathways and here we report the crystal structure of a fifth enzyme; the recently discovered amidinotransferase QueF‐Like (QueF‐L), responsible for the final step in the biosynthesis of archaeosine in the D‐loop of tRNA in a subset of Crenarchaeota. QueF‐L catalyzes the conversion of the nitrile group of the 7‐cyano‐7‐deazaguanine (preQ₀) base of preQ₀‐modified tRNA to a formamidino group. The structure, determined in the presence of preQ₀, reveals a symmetric T‐fold homodecamer of two head‐to‐head facing pentameric subunits, with 10 active sites at the inter‐monomer interfaces. Bound preQ₀ forms a stable covalent thioimide bond with a conserved active site cysteine similar to the intermediate previously observed in the nitrile reductase QueF. Despite distinct catalytic functions, phylogenetic distributions, and only 19% sequence identity, the two enzymes share a common preQ₀ binding pocket, and likely a common mechanism of thioimide formation. However, due to tight twisting of its decamer, QueF‐L lacks the NADPH binding site present in QueF. A large positively charged molecular surface and a docking model suggest simultaneous binding of multiple tRNA molecules and structure‐specific recognition of the D‐loop by a surface groove. The structure sheds light on the mechanism of nitrile amidation, and the evolution of diverse chemistries in a common fold. Proteins 2016; 85:103–116. © 2016 Wiley Periodicals, Inc.     

Activation of several tRNAs of Escherichia coli by the phenoxyacetyl derivative of N-hydroxysuccinimide

Escherichia coli 15T^? treated with chloramphenicol produces tRNA^phe which is deficient in minor nucleosides. Undermodified tRNA^phe chromatographs as two new peaks from a benzoylated diethylaminoethyl-cellulose column. Chloramphenicol tRNA^phe was purified by phenoxyacetylation of phenylalanyl-tRNA and subsequent chromatography on benzoylated diethylaminoethyl-cellulose. Purified tRNA^phe had an altered Chromatographie profile as a result of the purification procedure. Phenoxyacetylation of an unpurified tRNA preparation, which was either charged with phenylalanine or kept discharged, resulted in a permanent alteration of tRNA^phe which was similar to the alteration of the purified tRNA^phe. The altered tRNAs eluted with higher salt or ethanol concentrations from benzoylated diethylaminoethyl-cellulose. The alteration was also shown for tRNA^phe of phenoxyacetylated tRNA from late log phase E. coli 15T?. tRNA^glu and tRNA^Leu were not changed, but both tRNA^Arg and tRNA^Ile were altered. tRNA₂^Val and tRNA^Met shifted in the elution profile; tRNA₁^Val and tRNA_f^Met were not affected.Comparison of the primary structures of the alterable and nonalterable tRNA's revealed that all alterable tRNA's have the undefined nucleoside X in the extra loop. Phenoxyacetylation of nucleoside X probably was the cause of the altered profiles.tRNA^phe from E. coli 15T^? treated with chloramphenicol was less reactive towards phenoxyacetylation than normal tRNA, possibly because of a different conformation of the modification-deficient molecule relative to the normal tRNA^phe. tRNA^phe from E. coli 15T^?, starved for cysteine and methionine and treated with chloram-phenicol, is more deficient in minor nucleosides and showed even less reactivity.Acceptor capacities of the altered tRNA species were not changed significantly; only the acceptor capacity for tRNA^Ile decreased approximately 25%. The recognition site for the aminoacyl-tRNA synthetases probably is not affected.     

Use of polymerase chain reaction to identify a leucyl tRNA in Streptomyces coelicolor

Polymerase chain reaction (PCR) was used to amplify a fragment of DNA encoding a tRNA that recognizes the abundant CUC leucine codon from the chromosome of Streptomyces coelicolor. Sequence analysis of the gene, designated leuU, indicated that it codes for a tRNA 88 nucleotides in length that shares 75% identity with the Escherichia coli tRNA^Leu_CUC, while it shares only 65% identity with the only other sequenced leucyl tRNA from S. coelicolor, the bldA encoded tRNA^Leu_UUA. Accumulation of the leuU tRNA was examined by Northern blot analysis and shown to be present at constant levels throughout growth in contrast to the bldA-encoded tRNA which shows a temporal pattern of accumulation [Leskiw et al., 1993. J. Bacteriol., 175, 1995–2005].     

Plant mitochondria use two pathways for the biogenesis of tRNAHis

All tRNA^His possess an essential extra G_–1 guanosine residue at their 5′ end. In eukaryotes after standard processing by RNase P, G_–1 is added by a tRNA^His guanylyl transferase. In prokaryotes, G_–1 is genome-encoded and retained during maturation. In plant mitochondria, although trnH genes possess a G_–1 we find here that both maturation pathways can be used. Indeed, tRNA^His with or without a G_–1 are found in a plant mitochondrial tRNA fraction. Furthermore, a recombinant Arabidopsis mitochondrial RNase P can cleave tRNA^His precursors at both positions G₊₁ and G_–1. The G_–1 is essential for recognition by plant mitochondrial histidyl-tRNA synthetase. Whether, as shown in prokaryotes and eukaryotes, the presence of uncharged tRNA^His without G_–1 has a function or not in plant mitochondrial gene regulation is an open question. We find that when a mutated version of a plant mitochondrial trnH gene containing no encoded extra G is introduced and expressed into isolated potato mitochondria, mature tRNA^His with a G_–1 are recovered. This shows that a previously unreported tRNA^His guanylyltransferase activity is present in plant mitochondria.     

Cytokinin-Active Ribonucleosides in Phaseolus RNA: II. DISTRIBUTION IN tRNA SPECIES FROM ETIOLATED P. VULGARIS L. SEEDLINGS

The distribution of cytokinin-active ribonucleosides in tRNA species from etiolated Phaseolus vulgaris L. seedlings has been examined. Phaseolus tRNA was fractionated by benzoylated diethylaminoethyl-cellulose and RPC-5 chromatography, and the distribution of cytokinin activity was compared with the distribution of tRNA species expected to correspond to codons beginning with U. Phaseolus tRNA^Cys, tRNA^Trp, tRNA^Tyr, a major peak of tRNA^Phe, and a large fraction of tRNA^Leu were devoid of cytokinin activity in the tobacco bioassay. Cytokinin activity was associated with all fractions containing tRNA^Ser species and with minor tRNA^Leu species. In addition, several anomalous peaks of cytokinin activity that could not be directly attributed to U group tRNA species were detected.     

Overproduction and purification of Escherichia coli tRNALeu

Chemically synthesized genes encodingEscherichia coli tRNA ₁ ^Leu and tRNA ₂ ^Leu were ligated into the plasmid pTrc99B. then transformed intoEscherichia coli MT102, respectively. The positive transformants, named MT-Leu1 and MT-Leu2, were confirmed by DNA sequencing, and the conditions of cultivation for the two transformants were optimized. As a result, leucinc accepting activity of their total tRNA reached 810 and 560 pmol/A₂₆₀, respectively: the content of tRNA ₁ ^Leu was 50% of total tRNA from MT-Leu1, while that of tRNA ₂ ^Leu was 30% of total tRNA from MT-Leu2. Both tRNA^Leus from their rotal tRNs were fractionated to 1 600 pmol/A₂₆₀ after DEAE-Sepharose and BD-cellulose column chromatography. The accurate kinetic constants of aminoacylation of the two isoacceptors of tRNA^Leu catalyzed by leucyl-tRNA synthetase were determined. Project supported by the National Natural Science Foundation of China (Grant No. 39570164).     

Distribution of Cytokinin-active Ribonucleosides in Wheat Germ tRNA Species

The distribution of cytokinin activity in wheat (Triticum aestivum) germ tRNA fractionated by BD-cellulose and RPC-5 chromatography has been examined. As in other organisms, the cytokinin moieties in wheat germ tRNA appear to be restricted to tRNA species that would be expected to respond to codons beginning with U. Only a few of the wheat germ tRNA species in this coding group actually contain cytokinin modifications. Cytokinin activity was associated with isoaccepting tRNA^Ser species and with a minor tRNA^Leu species from wheat germ. All other wheat germ tRNA species corresponding to codons beginning with U were devoid of cytokinin activity in the tobacco callus bioassay.     

The yeast cloning vector YEp13 contains a tRNA3Leu gene that can mutate to an amber suppressor

We have shown that the yeast-Escherichia coli shuttle vector YEp 13 contains, as part of its yeast chromosomal segment, a tRNA₃^Leu gene. We have also isolated and characterized a variant of YEp13, namely YEp13-a, which is capable of suppressing a variety of yeast amber-suppressible alleles in vivo. YEp13-a differs from YEp13 by a single point mutation, which changes the three-nucleotide, plus-strand sequence corresponding to the tRNA₃^Leu anticodon from the normal C-A-A to C-T-A. This nucleotide change creates a site for the restriction enzyme XbaI in the suppressor tRNA₃^Leu gene. We have taken advantage of the correlation between the suppressor mutation and the XbaI site formation, to show that the tRNA₃^Leu gene on YEp13 corresponds to the genetically characterized yeast chromosomal amber suppressor SUP53. We have also shown that SUP53 is located just centromere-distal to LEU2 on chromosome III. Finally, comparison of the DNA sequence of SUP53 and its flanking regions with the sequences of other cloned yeast tRNA₃^Leu genes has revealed considerable sequence homology in the immediate 5′-flanking regions of these genes.     

The determination of tRNALeu recognition nucleotides for Escherichia coli L/F transferase

Escherichia coli leucyl/phenylalanyl-tRNA protein transferase catalyzes the tRNA-dependent post-translational addition of amino acids onto the N-terminus of a protein polypeptide substrate. Based on biochemical and structural studies, the current tRNA recognition model by L/F transferase involves the identity of the 3′ aminoacyl adenosine and the sequence-independent docking of the D-stem of an aminoacyl-tRNA to the positively charged cluster on L/F transferase. However, this model does not explain the isoacceptor preference observed 40 yr ago. Using in vitro-transcribed tRNA and quantitative MALDI-ToF MS enzyme activity assays, we have confirmed that, indeed, there is a strong preference for the most abundant leucyl-tRNA, tRNA^Leu (anticodon 5′-CAG-3′) isoacceptor for L/F transferase activity. We further investigate the molecular mechanism for this preference using hybrid tRNA constructs. We identified two independent sequence elements in the acceptor stem of tRNA^Leu (CAG)—a G₃:C₇₀ base pair and a set of 4 nt (C₇₂, A₄:U₆₉, C₆₈)—that are important for the optimal binding and catalysis by L/F transferase. This maps a more specific, sequence-dependent tRNA recognition model of L/F transferase than previously proposed.     

Interactions of yeast valyl-tRNA synthetase with RNAs and conformational changes of the enzyme.

Different conformations have been identified for the enzyme valyl-tRNA synthetase from yeast inside its complex with one tRNA molecule by neutron scattering. One form is identical to that of the free enzyme in solution; the other form is more contracted, having a radius of gyration which is smaller by 10% and a specific volume which is smaller by 1%. The contracted conformation has been found for the complexes with tRNA^Val and tRNA^Asp in phosphate buffer (pH 6.3) provided the ionic strength is lower than about 150 mm. In higher ionic strength (up to about 500 mm) the enzyme still forms a complex with tRNA^Val but its conformation remains that of the free protein in solution. In the complex with tRNA₃^Leu, the enzyme conformation is that of the free state even at the lowest ionic strength examined (that of the phosphate buffer, 60 mm). The free enzyme is an elongated molecule of radius of gyration 40 Å (a compact protein of the same molecular weight would have a radius of gyration of 30 Å).The positioning within the complex of tRNA^Val, on the one hand, and tRNA₃^Leu, on the other, is very different. The first tRNA is intimately associated with the enzyme, lying predominantly closer to the centre of mass of the complex than the protein. In the complex with tRNA₃^Leu, the tRNA lies further away from the centre of mass of the complex than the protein.Small concentrations of tRNA^Val, tRNA^Asp, tRNA₃^Leu or Escherichia coli 5 S ribosomal RNA cause the enzyme to aggregate into dimers, trimers and higher aggregates provided the ionic strength of the buffer is below 150 mm. In higher ionic strength or for [RNA]: [enzyme] > 1 the aggregates are dissociated to yield the one-to-one RNA-enzyme complex.