Enzymatic binding of aminoacyl transfer ribonucleic acid to ribosomes: the study of binding sites of 2' and 3' isomers of aminoacyl transfer ribonucleic acid.

The mechanism of enzymatic binding of AAtRNA to the acceptor site Escherichia coli ribosomes has been studied using the following aminoacyl oligonucleotides as models of the 3' terminus of AA-tRNA: C-A-Phe, C-A-(2'-Phe)H, and C-A(2'H)Phe. T-psi-C-Gp was used as a model of loop IV of tRNA. The EF-T dependent binding of Phe-tRNA to ribosomes (in the presence of either GTP or GMPPCP) and the GTPase activity associated with EF-T dependent binding of the Phe-tRNA were inhibited by C-A-Phe,C-A(2'Phe)H, and C-A(2'H)Phe. These aminoacyl oligonucleotides inhibit both the formation of ternary complex EF-Tu-GTP-AA-tRNA and the interaction of this complex with the ribosomal A site. The uncoupled EF-Tu dependent GTPase (in the absence of AA-tRNA) was also inhibited by C-A-Phe, C-A(2'Phe)H, and C-A(2'H)Phe, while nonenzymatic binding of Phe-tRNA to the ribosomal A site was inhibited by C-A-Phe and C-A(2'-Phe)H, but not by C-A(2'H)Phe. The tetranucleotide T-psi-C-Gp inhibited both enzyme binding of Phe-tRNA and EF-T dependent GTP hydrolysis. However, inhibition of the latter reaction occured at a lower concentration of T-psi-C-Gp suggesting a specific role of T-psi-C-Gp loop of AA-tRNA in the GTPase reaction. The role of the 2' and 3' isomers of AA-tRNA during enzymatic binding to ribosomes is discussed and it is suggested that 2' leads to 3' transacylation in AA-tRNA is a step which follows GTP hydrolysis but precedes peptide bond formation.     

Conformational changes of L-rRNA during elongation of polypeptide

A ribosome undergoes significant conformational changes during elongation of a polypeptide chain, and these are correlated with structural changes of rRNAs. We tested 15 different oligonucleotides complementary to the selected, highly conserved seqences of rRNAs (L-rRNA, 5S rRNA and tRNA) important in protein biosynthesis. We carried out a reaction of binding Phe-tRNA to A site and a polymerization of polypeptide chains on the ribosomes converted either to pre- or to posttranslocational states. The inhibition of polymerization reaction by complementary oligonucleotides was high in all ribosomal states. The efficiency of inhibition of binding reaction was lower and more diverse than was the polypeptide elongation. We conclude that the selected oligonucleotides inhibit polypeptide synthesis with different effectivity, primarily depending on L-rRNA conformation within ribosome architecture.     

Two tRNA-binding sites in addition to A and P sites on eukaryotic ribosomes.

The interaction of tRNA with 80 S ribosomes from rabbit liver was studied using biochemical as well as fluorescence techniques. Besides the canonical A and P sites, two additional sites were found which specifically bind deacylated tRNA. One of the sites is analogous to the E site of prokaryotic ribosomes, in that binding of tRNA is labile, does not depend on codon-anticodon interaction, does not protect the anticodon loop from solvent access, and requires the presence of the 3'-terminal adenosine of the tRNA. In contrast, the stability of the tRNA complex with the second site (S site) is high. tRNA binding to the S site is also codon-independent; nevertheless, the anticodon loop is shielded from solvent access. Removal of the 3'-terminal adenosine decreases the affinity of tRNA(Phe) for the S site approximately 50-fold. tRNA(Phe) is retained at the S site during translocation and through poly(Phe) synthesis. Thus, the S site does not seem to be an intermediate site for the tRNA during the elongation cycle. Rather, the tRNA bound to the S site may allosterically modulate the function of the ribosome.     

The effect of modification of nucleotide-37 on the interaction of aminoacyl-tRNA with the A-site of the 70S ribosome

To estimate the effect of modified nucleotide-37, the interaction of two yeast aminoacyl-tRNAs (Phe-tRNAK+YPhe and Phe-tRNAK-YPhe) with the A site of complex [70S.poly(U).deacylated tRNA(Phe) in the P site] was assayed at 0-20 degrees C. As comparisons with native Phe-tRNAK+YPhe showed, removal of the Y base decreased the association constant of Phe-tRNAK-YPhe and the complex by an order of magnitude at any temperature, and increased the enthalpy of their interaction by 23 kJ/mol. When the Y base was present in the anticodon loop of deacylated tRNA(Phe) bound to the P site of the 70S ribosome, twice higher affinity for the A site was observed for Phe-tRNAK-YPhe but not for Phe-tRNAK+YPhe. Thus, the modified nucleotide 3' of the Phe-tRNA(Phe) anticodon stabilized the codon-anticodon interaction both in the A and in the P sites of the 70S ribosome.     

Function of Modified Nucleosides 7-Methylguanosine, Ribothymidine, and 2-Thiomethyl-N6-(Isopentenyl)adenosine in Procaryotic Transfer Ribonucleic Acid

To elucidate subtle functions of transfer ribonucleic acid (tRNA) modifications in protein synthesis, pairs of tRNA's that differ in modifications at specific positions were prepared from Bacillus subtilis. The tRNA's differ in modifications in the anticodon loop, the extra arm, and the TUC loop. The functional properties of these species were compared in aminoacylation, as well as in initiation and peptide bond formation, at programmed ribosomes. These experiments demonstrated the following. (i) In tRNA(f) (Met) the methylation of guanosine 46 in the extra arm to 7-methylguanosine by the 7-methylguanosine-forming enzyme from Escherichia coli changes the aminoacylation kinetics for the B. subtilis methionyl-tRNA synthetase. In repeated experiments the V(max) value is decreased by one-half. (ii) tRNA(f) (Met) species with ribothymidine at position 54 (rT54) or uridine at position 54 (U54) were obtained from untreated or trimethoprim-treated B. subtilis. The formylated fMet-tRNA(f) (Met) species with U54 and rT54, respectively, function equally well in an in vitro initiation system containing AUG, initiation factors, and 70s ribosomes. The unformylated Met-tRNA(t) (Met) species, however, differ from each other: "Met-tRNA(f) (Met) rT" is inactive, whereas the U54 counter-upart effectively forms the initiation complex. (iii) Two isoacceptors, tRNA(1) (Phe) and tRNA(2) (Phe), were obtained from B. subtilis. tRNA(1) (Phe) accumulates only under special growth conditions and is an incompletely modified precursor oftRNA(2) (Phe): in the first position of the anticodon, guanosine replaces Gm, and next to the 3' end of the anticodon (isopentenyl)adenosine replaces 2-thiomethyl-N(6)-(isopentenyl)adenosine. Both tRNA's behave identically in aminoacylation kinetics. In the factor-dependent AUGU(3)-directed formation of fMet-Phe, the undermodified tRNA(1) (Phe) is always less efficient at Mg(2+) concentrations between 5 and 15 mM than its mature counterpart.     

Identification of 2'-hydroxyl groups required for interaction of a tRNA anticodon stem-loop region with the ribosome.

Synthetic RNA stem loops corresponding to positions 28-42 in the anticodon region of tRNA(Phe) bind efficiently in an mRNA-dependent manner to ribosomes, whereas those made from DNA do not. In order to identify the positions where ribose is required, the anticodon stem-loop region of tRNA(Phe) (Escherichia coli) was synthesized chemically using a mixture of 2'-hydroxyl- and 2'-deoxynucleotide phosphoramidites. Oligonucleotides whose ribose composition allowed binding were retained selectively on nitrocellulose filters via binding to 30S ribosomal subunits. The binding-competent oligonucleotides were submitted to partial alkaline hydrolysis to identify the positions that were enriched for ribose. Quantification revealed a strong preference for a 2'-hydroxyl group at position U33. This was shown directly by the 50-fold lower binding affinity of a stem loop containing a single deoxyribose at position U33. Similarly, defective binding of the corresponding U33-2'-O-methyl-substituted stem-loop RNA suggests that absence of the 2'-hydroxyl group, rather than an altered sugar pucker, is responsible. Stem-loop oligoribonucleotides from different tRNAs with U33-deoxy substitutions showed similar, although quantitatively different effects, suggesting that intramolecular rather than tRNA-ribosome interactions are affected. Because the 2'-hydroxyl group of U33 was shown to be a major determinant of the U-turn of the anticodon loop in the crystal structure of tRNA(Phe) in yeast, our finding might indicate that the U-turn conformation in the anticodon loop is required and/or maintained when the tRNA is bound to the ribosomal P site.     

Phenylalanyl-tRNA synthetase of Escherichia coli K 10. Multiple enzyme-aminoacyl-tRNA complexes as a consequence of substrate specificity.

The interaction between Phe-tRNA(Phe) or other acyl-tRNA derivatives thereof and phenylalanyl-tRNA synthetase of Escherichia coli K 10 has been investigated by nonequilibrium dialysis, by fluorescence titration in the presence of 2-p-toluidinylnaphthalene-6-sulfonate, by the kinetics of the aminoacylation of tRNA(Phe), and by the kinetics of the catalytic hydrolysis of Phe-tRNA(Phe). Phe-tRNA(Phe), or derivatives thereof, forms two types of complexes with the synthetase. One type involves the attachment of the phenylalanyl moiety to the phenylalanine-specific site of the enzyme, and the other type, to the tRNA(Phe)-specific binding site. They resemble alternative modes of a destabilized enzyme-product complex and are predicted on the basis of thermodynamic considerations. The two modes of binding of acyl-tRNA compete with each other. The attachment of Phe-tRNA(Phe) to the phenylalanine-specific site dominates. At equilibrium, this complex is present at a fourfold higher concentration than the other type of complex. The HNO2 deaminated Phe-tRNA(Phe) binds exclusively to the site specific for L-phenylalanine. On the contrary, Ile-tRNA(Phe) adds at 94.1% to the tRNA(Phe)-specific site. The association of Phe-tRNA(Phe) with this site leads to enzymatic hydrolysis into L-phenylalanine and tRNA(Phe). The complex involving the phenylalanine-specific site is hydrolytically unproductive. L-Phenylalanine acts as an activator of the hydrolysis by occupying the amino acid specific site and by shifting the equilibrium between the complexes toward the binding ot Phe-tRNA(Phe) at the tRNA(Phe)-specific site. The association of Phe-tRNA(Phe) at the phenylalanine-specific site does not interfere sterically with the binding of free tRNA(Phe). The sequential addition of free and aminoacylated tRNA(Phe) exhibits negative cooperativity. Such a mechanism could help to expel the product from the enzyme.     

Interaction of human and Escherichia coli tRNA(Phe) with human 80S ribosomes in the presence of oligo- and polyuridylate templates.

Human placenta and Escherichia coli Phe-tRNA(Phe) and N-AcPhe-tRNA(Phe) binding to human placenta 80S ribosomes was studied at 13 mM Mg2+ and 20 degrees C in the presence of poly(U), (pU)6 or without a template. Binding properties of both tRNA species were studied. Poly(U)-programmed 80S ribosomes were able to bind charged tRNA at A and P sites simultaneously under saturating conditions resulting in effective dipeptide formation in the case of Phe-tRNA(Phe). Affinities of both forms of tRNA(Phe) to the P site were similar (about 1 x 10(7) M-1) and exceeded those to the A site. Affinity of the deacylated tRNA(Phe) to the P site was much higher (association constant > 10(10) M-1). Binding at the E site (introduced into the 80S ribosome by its 60S subunit) was specific for deacylated tRNA(Phe). The association constant of this tRNA to the E site when A and P sites were preoccupied with N-AcPhe-tRNA(Phe) was estimated as (1.7 +/- 0.1) x 10(6) M-1. In the presence of (pU)6, charged tRNA(Phe) bound loosely at the A and P sites, and the transpeptidation level exceeded the binding level due to the exchange with free tRNA from solution. Affinities of aminoacyl-tRNA to the A and P sites in the presence of (pU)6 seem to be the same and much lower than those in the case of poly(U). Without a messenger, binding of the charged tRNA(Phe) to 80S ribosomes was undetectable, although an effective transpeptidation was observed suggesting a very labile binding of the tRNA simultaneously at the A and P sites.     

tRNA discrimination by T. thermophilus phenylalanyl-tRNA synthetase at the binding step

The extent of tRNA recognition at the level of binding by Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS), one of the most complex class II synthetases, has been studied by independent measurements of the enzyme association with wild-type and mutant tRNA(Phe)s as well as with non-cognate tRNAs. The data obtained, combined with kinetic data on aminoacylation, clearly show that PheRS exhibits more tRNA selectivity at the level of binding than at the level of catalysis. The anticodon nucleotides involved in base-specific interactions with the enzyme prevail both in the initial binding recognition and in favouring aminoacylation catalysis. Tertiary nucleotides of base pair G19-C56 and base triple U45-G10-C25 contribute primarily to stabilization of the correctly folded tRNA(Phe) structure, which is important for binding. Other nucleotides of the central core (U20, U16 and of the A26-G44 tertiary base pair) are involved in conformational adjustment of the tRNA upon its interaction with the enzyme. The specificity of nucleotide A73, mutation of which slightly reduces the catalytic rate of aminoacylation, is not displayed at the binding step. A few backbone-mediated contacts of PheRS with the acceptor and anticodon stems revealed in the crystal structure do not contribute to tRNA(Phe) discrimination, their role being limited to stabilization of the complex. The highest affinity of T. thermophilus PheRS for cognate tRNA, observed for synthetase-tRNA complexes, results in 100-3000-fold binding discrimination against non-cognate tRNAs.     

Phenylalanyl-tRNA synthetase contains a dispensable RNA-binding domain that contributes to the editing of noncognate aminoacyl-tRNA

Phenylalanyl-tRNA synthetase (PheRS) is a multidomain (alphabeta)2 heterotetrameric protein responsible for synthesizing Phe-tRNA(Phe) during protein synthesis. Previous studies showed that the alpha subunit forms the catalytic core of the enzyme, while the beta subunit contains a number of autonomous structural modules with a wide range of functions including tRNA anticodon binding and editing of the misaminoacylated species Tyr-tRNA(Phe). The B2 domain of the beta subunit is a structural homologue of the EMAPII/OB fold, which has been shown in other systems to contribute to tRNA binding. Structural studies of PheRS indicated that the B2 domain is distant from bound tRNA(Phe), leaving the role of this module in question. On the basis of homology modeling with other EMAPII domain-containing proteins, the 110 amino acid B2 domain was deleted to produce PheRS deltaB2. Full-length PheRS and PheRS deltaB2 showed comparable kinetics for in vitro aminoacylation, and both enzymes complemented a defect in phenylalanylation in vivo. PheRS deltaB2 showed a 2-fold drop compared to full-length PheRS in the catalytic efficiency (kcat/KM) of Tyr-tRNA(Phe) hydrolysis, suggesting a role for the B2 domain in post-transfer editing. A comparison of tRNA binding by full-length PheRS and PheRS deltaB2 indicated that the B2 domain acts as a secondary tRNA-binding site that could contribute to editing by promoting the translocation of mischarged tRNA to the editing site of PheRS. This proposed role for the B2 domain of PheRS is consistent with previous studies, suggesting that the highly conserved EMAPII fold is able to modulate the affinity of tRNA for its primary binding site.     

Apparent association constants of tRNAs for the ribosomal A, P, and E sites.

Association constants for tRNA binding to poly(U) programmed ribosomes were assessed under standardized conditions with a single preparation of ribosomes, tRNAs, and elongation factors, respectively, at 15 and 10 mM Mg2+. Association constants were determined by Scatchard plot analysis (the constants are given in units of [10(7)/M] measured at 15 mM Mg2+): the ternary complex Phe-tRNA.elongation factor EF-Tu.GTP (12 +/- 3), Phe-tRNA (1 +/- 0.4), AcPhe-tRNA (0.7 +/- 0.3), and deacylated tRNA(Phe) (0.4 +/- 0.15) bind with decreasing affinity to the A site of poly(U)-programmed ribosomes. tRNA(Phe) (7.2 +/- 0.8) binds to the P site with higher affinity than AcPhe-tRNA (3.7 +/- 1.3). The affinity of the E site for deacylated tRNA(Phe) (1 +/- 0.2) is about the same as that of the A site for AcPhe-tRNA (0.7 +/- 0.3). At lower Mg2+ concentrations the affinity of the E site ligand becomes stronger relative to the affinities of the A site ligands. Phe-tRNA and ternary complexes can occupy the A site at 0 degrees C in the presence of poly(U) even if the P site is free, whereas, as already known, deacylated tRNA or AcPhe-tRNA bind first to the P site of programmed ribosomes. Hill plot analyses of the binding data confirm an allosteric linkage between A and E sites in the sense of a negative cooperativity.     

Effect of ribosome binding and translocation on the anticodon of tRNAPhe as studied by wybutine fluorescence.

The complexes of N-AcPhe-tRNAPhe (or non-aminoacylated tRNAPhe) from yeast with 70S ribosomes from E. coli have been studied fluorimetrically utilizing wybutine, the fluorophore naturally occurring next to the 3' side of the anticodon, as a probe for conformational changes of the anticodon loop. The fluorescence parameters are very similar for tRNA bound to both ribosomal sites, thus excluding an appreciable conformational change of the anticodon loop upon translocation. The spectral change observed upon binding of tRNAPhe to the P site even in the absence of poly(U) is similar to the one brought about by binding of poly(U) alone to the tRNA. This effect may be due to a hydrophobic binding site of the anticodon loop or to a conformational change of the loop induced by binding interactions of various tRNA sites including the anticodon.     

Correlation of the structure and conformational changes of selected fragments of plant small ribosomal RNA within the steps of polypeptide chain elongation

The interaction and conformational relationships between rRNAs and ribosomal proteins are responsible for ribosome activity. We tested seven different deoxyoligonucleotides complementary to the selected, highly conserved sequences of 18S rRNAs important in protein biosynthesis. We carried out a reaction of binding Phe-tRNA to A site on the ribosomes converted either to pre- or to post-translocational states (with or without pre-hybridized oligonucleotides). We found a correlation between the level of oligomer hybridization and the inhibition of AA-tRNA binding. We observed well-defined structural changes of ribosome's conformation during different steps of the elongation cycle of protein biosynthesis.     

Role of modified nucleosides of yeast tRNAPhe in ribosomal binding

Naturally occurring nucleoside modifications are an intrinsic feature of transfer RNA (tRNA), and have been implicated in the efficiency, as well as accuracy-of codon recognition. The structural and functional contributions of the modified nucleosides in the yeast tRNA(Phe) anticodon domain were examined. Modified nucleosides were site-selectively incorporated, individually and in combinations, into the heptadecamer anticodon stem and loop domain, (ASL(Phe)). The stem modification, 5-methylcytidine, improved RNA thermal stability, but had a deleterious effect on ribosomal binding. In contrast, the loop modification, 1-methylguanosine, enhanced ribosome binding, but dramatically decreased thermal stability. With multiple modifications present, the global ASL stability was mostly the result of the individual contributions to the stem plus that to the loop. The effect of modification on ribosomal binding was not predictable from thermodynamic contributions or location in the stem or loop. With 4/5 modifications in the ASL, ribosomal binding was comparable to that of the unmodified ASL. Therefore, modifications of the yeast tRNA(Phe) anticodon domain may have more to do with accuracy of codon reading than with affinity of this tRNA for the ribosomal P-site. In addition, we have used the approach of site-selective incorporation of specific nucleoside modifications to identify 2'O-methylation of guanosine at wobble position 34 (Gm34) as being responsible for the characteristically enhanced chemical reactivity of C1400 in Escherichia coli 16S rRNA upon ribosomal footprinting of yeast tRNA(Phe). Thus, effective ribosome binding of tRNA(Phe) is a combination of anticodon stem stability and the correct architecture and dynamics of the anticodon loop. Correct tRNA binding to the ribosomal P-site probably includes interaction of Gm34 with 16S rRNA C1400.     

The role of the anticodon in the interaction between methionyl-tRNA synthetase and bacterial initiator tRNA?

Complementary and antiparallel oligonucleotides bind to exposed regions of the tRNA molecule. Aminoacylation in the presence of triplets has been used to determine the role of the anticodon in the interaction between methionyl-tRNA synthetase and initiator tRNA. ApUpG has no effect on the charging even when 70% of the tRNA is bound to the triplet, whereas in the presence of GpGpU which binds to the A-C-C sequence adjacent to the 3' terminal adenosine that fraction of the tRNA which is bound to the triplet is completely unavailable for charging. Hence the anticodon is probably not involved in a primary interaction while the A-C-C-A-OH clearly is. This conclusion is supported by the failure of the isolated anticodon loop and stem oligonucleotides to inhibit the aminoacylation reaction.     

Conformational change of the L-shaped tRNA(Phe) molecule

Fluorophore of proflavine was introduced onto the 3'-terminal ribose moiety of yeast tRNA(Phe). The distance between the fluorophore and the fluorescent Y base in the anticodon of yeast tRNA(Phe) was measured by a singlet-singlet energy transfer. Conformational changes of tRNA(Phe) with binding of tRNA(2Glu), which has the anticodon UUC complementary to the anticodon GAA of tRNA(Phe), were investigated. The distance obtained at the ionic strength of 100 mM K+ and 10 mM Mg2+ is very close to the distance from x-ray diffraction, while the distance obtained in the presence of tRNA(2Glu) is significantly smaller. Further, using a fluorescent probe of 4-bromomethyl-7-methoxycoumarin introduced onto pseudouridine residue psi 55 in the T psi C loop of tRNA(Phe), Stern-Volmer quenching experiments for the probe with or without added tRNA(2Glu) were carried out. The results showed greater access of the probe to the quencher with added tRNA(2Glu). These results suggest that both arms of the L-shaped tRNA structure tend to bend inside with binding of tRNA(2Glu) and some structural collapse occurs at the corner of the L-shaped structure.     

Transit of tRNA through the Escherichia coli ribosome: cross-linking of the 3' end of tRNA to ribosomal proteins at the P and E sites

Photoreactive derivatives of yeast tRNA(Phe) containing 2-azidoadenosine at their 3' termini were used to trace the movement of tRNA across the 50S subunit during its transit from the P site to the E site of the 70S ribosome. When bound to the P site of poly(U)-programmed ribosomes, deacylated tRNA(Phe), Phe-tRNA(Phe) and N-acetyl-Phe-tRNA(Phe) probes labeled protein L27 and two main sites within domain V of the 23S RNA. In contrast, deacylated tRNA(Phe) bound to the E site in the presence of poly(U) labeled protein L33 and a single site within domain V of the 23S rRNA. In the absence of poly(U), the deacylated tRNA(Phe) probe also labeled protein L1. Cross-linking experiments with vacant 70S ribosomes revealed that deacylated tRNA enters the P site through the E site, progressively labeling proteins L1, L33 and, finally, L27. In the course of this process, tRNA passes through the intermediate P/E binding state. These findings suggest that the transit of tRNA from the P site to the E site involves the same interactions, but in reverse order. Moreover, our results indicate that the final release of deacylated tRNA from the ribosome is mediated by the F site, for which protein L1 serves as a marker. The results also show that the precise placement of the acceptor end of tRNA on the 50S subunit at the P and E sites is influenced in subtle ways both by the presence of aminoacyl or peptidyl moieties and, more surprisingly, by the environment of the anticodon on the 30S subunit.     

tRNAHis guanylyltransferase adds G-1 to the 5' end of tRNAHis by recognition of the anticodon, one of several features unexpectedly shared with tRNA synthetases

All eukaryotic tRNA(His) molecules are unique among tRNA species because they require addition of a guanine nucleotide at the -1 position by tRNA(His) guanylyltransferase, encoded in yeast by THG1. This G(-1) residue is both necessary and sufficient for aminoacylation of tRNA by histidyl-tRNA synthetase in vitro and is required for aminoacylation in vivo. Although Thg1 is presumed to be highly specific for tRNA(His) to prevent misacylation of tRNAs, the source of this specificity is unknown. We show here that Thg1 is >10,000-fold more selective for its cognate substrate tRNA(His) than for the noncognate substrate tRNA(Phe). We also demonstrate that the GUG anticodon of tRNA(His) is a crucial Thg1 identity element, since alteration of this anticodon in tRNA(His) completely abrogates Thg1 activity, and the simple introduction of this GUG anticodon to any of three noncognate tRNAs results in significant Thg1 activity. For tRNA(Phe), k(cat)/K(M) is improved by at least 200-fold. Thg1 is the only protein other than aminoacyl-tRNA synthetases that is known to use the anticodon as an identity element to discriminate among tRNA species while acting at a remote site on the tRNA, an unexpected link given the lack of any identifiable sequence similarity between these two families of proteins. Moreover, Thg1 and tRNA synthetases share two other features: They act in close proximity to one another at the top of the tRNA aminoacyl-acceptor stem, and the chemistry of their respective reactions is strikingly similar.