Role of residue Glu152 in the discrimination between transfer RNAs by tyrosyl-tRNA synthetase from Bacillus stearothermophilus.

Residue Glu152 of tyrosyl-tRNA synthetase (TyrTS) from Bacillus stearothermophilus is close to phosphate groups 73 and 74 of tRNATyr in the structural model of their complex. TyrTS(E152A), a mutant synthetase carrying the change of Glu152 to Ala, was toxic when overproduced in Escherichia coli. The toxicity strongly increased with the growth temperature. It was measured by the ratios of the efficiencies with which the producing cells plated in induced or repressed conditions and at 30 degrees C or 37 degrees C. TyrTS(E152Q), TyrTS(E152D) and the wild-type synthetase were not toxic in conditions where TyrTS(E152A) was toxic. The toxicity of TyrTS(E152A) was abolished by additional mutations of the synthetase that prevent the binding of tRNATyr but not by a mutation that prevents the formation of Tyr-AMP. Because TyrTS(E152A) was active for the aminoacylation of tRNATyr, its toxicity could only be due to faulty interactions with non-cognate tRNAs, either their non-productive binding or their mischarging with tyrosine. TyrTS(E152A) and TyrTS(E152Q) mischarged tRNAPhe and tRNAVal in vitro with tyrosine unlike TyrTS(E152D) or the wild-type enzyme. Thus, several features of the side-chain in position 152 of TyrTS, including its negative charge, are important for the rejection of non-cognate tRNAs. TyrTS(E152A), TyrTS(E152D) and TyrTS(E152Q) had similar steady-state kinetics parameters for the charging of tRNATyr with tyrosine in vitro, with kcat/KM ratios improved 2.5 times relative to the wild-type synthetase. We conclude that the side-chain of residue Glu152 weakens the binding of TyrTS to tRNATyr and prevents its interaction with non-cognate tRNAs.     

Evidence for two types of complexes formed by yeast tyrosyl-tRNA synthetase with cognate and non-cognate tRNA. Effect of ribonucleoside triphosphates

Polyacrylamide gel electrophoresis at pH 8.3 was used to detect and quantitate the formation of the yeast tyrosyl-tRNA synthetase (an alpha 2-type enzyme) complex with its cognate tRNA. Electrophoretic mobility of the complex is intermediate between the free enzyme and free tRNA; picomolar quantities can be readily detected by silver staining and quantitated by densitometry of autoradiograms when [32P]tRNA is used. Two kinds of complexes of Tyr-tRNA synthetase with yeast tRNA(Tyr) were detected. A slower-moving complex is formed at ratios of tRNA(Tyr)/enzyme less than or equal to 0.5; it is assigned the composition tRNA.(alpha 2)2. At higher ratios, a faster-moving complex is formed, approaching saturation at tRNA(Tyr)/enzyme = 1; any excess of tRNA(Tyr) remains unbound. This complex is assigned the composition tRNA.alpha 2. The slower, i.e. tRNA.(alpha 2)2 complex, but not the faster complex, can be formed even with non-cognate tRNAs. Competition experiments show that the affinity of the enzyme towards tRNA(Tyr) is at least 10-fold higher than that for the non-cognate tRNAs. ATP and GTP affect the electrophoretic mobility of the enzyme and prevent the formation of tRNA.(alpha 2)2 complexes both with cognate and non-cognate tRNAs, while neither tyrosine, as the third substrate of Tyr tRNA synthetase, nor AMP, AMP/PPi, or spermidine, have such effects. Hence, the ATP-mediated formation of the alpha 2 structure parallels the increase in specificity of the enzyme towards its cognate tRNA.     

Structural and kinetic bases for the recognition of tRNATyr by tyrosyl-tRNA synthetase

The aminoacylation of transfer RNA is a key step of translation since it relates amino acids to anticodons. To understand how the tyrosyl-tRNA synthetase (TyrTS) from Bacillus stearothermophilus recognizes tRNA(Tyr), we constructed 14 new mutant TyrTS by site-directed mutagenesis, determined their kinetic properties and used these and previous data to construct a detailed structural model of the complex between TyrTS and the acceptor arm of tRNA(Tyr). In the model Arg207, Lys208, Asn 146 and Glu 152 interact with phosphate groups. A contact between guanine 1 and Trp 196 is unspecific. Adenine 73, the fourth base from the 3' end, is specifically recognized through Trp 196 and the main-chain carbonyl of Ala150. At the active site, adenine 76 might interact with Lys82 and Arg86. There is a tight complementarity in shape between the tRNA and the synthetase. TyrTS and tRNA(Tyr) form an additional contact, in the vicinity of adenine 73, when their complex goes from the initial state to the transition state. The rate of aminoacylation, through the precise recognition of adenine 73, could thus be an important factor of discrimination by TyrTS among tRNAs.     

Competition of aminoacyl-tRNA synthetases for tRNA ensures the accuracy of aminoacylation.

The accuracy of protein biosynthesis rests on the high fidelity with which aminoacyl-tRNA synthetases discriminate between tRNAs. Correct aminoacylation depends not only on identity elements (nucleotides in certain positions) in tRNA (1), but also on competition between different synthetases for a given tRNA (2). Here we describe in vivo and in vitro experiments which demonstrate how variations in the levels of synthetases and tRNA affect the accuracy of aminoacylation. We show in vivo that concurrent overexpression of Escherichia coli tyrosyl-tRNA synthetase abolishes misacylation of supF tRNA(Tyr) with glutamine in vivo by overproduced glutaminyl-tRNA synthetase. In an in vitro competition assay, we have confirmed that the overproduction mischarging phenomenon observed in vivo is due to competition between the synthetases at the level of aminoacylation. Likewise, we have been able to examine the role competition plays in the identity of a non-suppressor tRNA of ambiguous identity, tRNA(Glu). Finally, with this assay, we show that the identity of a tRNA and the accuracy with which it is recognized depend on the relative affinities of the synthetases for the tRNA. The in vitro competition assay represents a general method of obtaining qualitative information on tRNA identity in a competitive environment (usually only found in vivo) during a defined step in protein biosynthesis, aminoacylation. In addition, we show that the discriminator base (position 73) and the first base of the anticodon are important for recognition by E. coli tyrosyl-tRNA synthetase.     

A bacterial amber suppressor in Saccharomyces cerevisiae is selectively recognized by a bacterial aminoacyl-tRNA synthetase.

Little is known about the conservation of determinants for the identities of tRNAs between organisms. We showed previously that Escherichia coli tyrosine tRNA synthetase can charge the Saccharomyces cerevisiae mitochondrial tyrosine tRNA in vivo, even though there are substantial sequence differences between the yeast mitochondrial and bacterial tRNAs. The S. cerevisiae cytoplasmic tyrosine tRNA differs in sequence from both its yeast mitochondrial and E. coli counterparts. To test whether the yeast cytoplasmic tyrosyl-tRNA synthetase recognizes the E. coli tRNA, we expressed various amounts of an E. coli tyrosine tRNA amber suppressor in S. cerevisiae. The bacterial tRNA did not suppress any of three yeast amber alleles, suggesting that the yeast enzymes retain high specificity in vivo for their homologous tRNAs. Moreover, the nucleotides in the sequence of the E. coli suppressor that are not shared with the yeast cytoplasmic tyrosine tRNA do not create determinants which are efficiently recognized by other yeast charging enzymes. Therefore, at least some of the determinants that influence in vivo recognition of the tyrosine tRNA are specific to the cell compartment and organism. In contrast, expression of the cognate bacterial tyrosyl-tRNA synthetase together with the bacterial suppressor tRNA led to suppression of all three amber alleles. The bacterial enzyme recognized its substrate in vivo, even when the amount of bacterial tRNA was less than about 0.05% of that of the total cytoplasmic tRNA.     

Evidence for unfolding of the single-stranded GCCA 3'-End of a tRNA on its aminoacyl-tRNA synthetase from a stacked helical to a foldback conformation

The conformation of a tRNA in its initial contact with its cognate aminoacyl-tRNA synthetase was investigated with the Escherichia coli glutamyl-tRNA synthetase-tRNA(Glu) complex. Covalent complexes between the periodate-oxidized tRNA(Glu) and its synthetase were obtained. These complexes are specific since none were formed with any other oxidized E. coli tRNA. The three major residues cross-linked to the 3'-terminal adenosine of oxidized tRNA(Glu) are Lys115, Arg209, and Arg48. Modeling of the tRNA(Glu)-glutamyl-tRNA synthetase based on the known crystal structures of Thermus thermophilus GluRS and of the E. coli tRNA(Gln)-glutaminyl-tRNA synthetase complex shows that these three residues are located in the pocket that binds the acceptor stem, and that Lys115, located in a 26 residue loop closed by coordination to a zinc atom in the tRNA acceptor stem-binding domain, is the first contact point of the 3'-terminal adenosine of tRNA(Glu). In our model, we assume that the 3'-terminal GCCA single-stranded segment of tRNA(Glu) is helical and extends the stacking of the acceptor stem. This assumption is supported by the fact that the 3' CCA sequence of tRNA(Glu) is not readily circularized in the presence of T4 RNA ligase under conditions where several other tRNAs are circularized. The two other cross-linked sites are interpreted as the contact sites of the 3'-terminal ribose on the enzyme during the unfolding and movement of the 3'-terminal GCCA segment to position the acceptor ribose in the catalytic site for aminoacylation.     

Mutational switching of a yeast tRNA synthetase into a mammalian-like synthetase cytokine

Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs. A link was recently established between protein biosynthesis and cytokine signal transduction. Human tyrosyl-tRNA synthetase can be split into two fragments, each of which has a distinct cytokine function. This activity is specific to the human enzyme. It is absent in the enzymes from lower organisms such as bacteria and yeast. Here, yeast tyrosyl-tRNA synthetase (TyrRS), which lacks cytokine activity, was used as a model to explore how a human tyrosyl-tRNA synthetase during evolution acquired novel functions beyond aminoacylation. We found that a rationally designed mutant yeast TyrRS(ELR) gained cytokine function. The mutant yeast enzyme gained this function without sacrifice of aminoacylation activity. Therefore, relatively simple alteration of a basic structural motif imparts cytokine activity to a tRNA synthetase while preserving its canonical function. Further work established that mutational switching of a yeast protein to a mammalian-like cytokine was specific to this synthetase and not to just any yeast ortholog of a mammalian cytokine.     

Altered expression of plant lysyl tRNA synthetase promotes tRNA misacylation and translational recoding of lysine

The Arabidopsis thaliana lysyl tRNA synthetase (AtKRS) structurally and functionally resembles the well-characterized prokaryotic class IIb KRS, including the propensity to aminoacylate tRNA(Lys) with suboptimal identity elements, as well as non-cognate tRNAs. Transient expression of AtKRS in carrot cells promotes aminoacylation of such tRNAs in vivo and translational recoding of lysine at nonsense codons. Stable expression of AtKRS in Zea mays causes translational recoding of lysine into zeins, significantly enriching the lysine content of grain.     

An E. coli aminoacyl-tRNA synthetase can substitute for yeast mitochondrial enzyme function in vivo

We have investigated the function of an E. coli aminoacyl-tRNA synthetase in S. cerevisiae strains that are respiration-deficient because of a mutation or a gene disruption in the nuclear encoded gene for the mitochondrial tyrosyl-tRNA synthetase. Although the yeast mitochondrial and E. coli tyrosine tRNAs differ significantly in sequence, expression of the E. coli tyrosyl-tRNA synthetase from a gene fusion restores respiration. The fusion gene contains a presumptive sequence for mitochondrial import from the mitochondrial tyrosyl-tRNA synthetase gene fused to the E. coli coding region. The fusion protein is incorporated into mitochondria. This incorporation and the rescue of the respiratory defect require the presumptive sequence for mitochondrial import. These experiments suggest a more limited definition of the identity of a tyrosine tRNA.     

Recognition between tRNAs and aminoacyl-tRNA-synthetases of different specificities]

A few examples of incorrect interactions between aminoacyl-tRNA-synthetases and tRNAs extracted from the same organism have already been demonstrated. These interactions can lead, in most cases, to incorrect aminoacylations. The lack of specificity of the aminoacyl-tRNA suggests that incorrect interactions could be a general phenomenon. The aim of this study is to check whether incorrect interactions are a general feature, i.e. whether every aminoacyl-tTNA-synthetase is able to interact with homologous non-cognate tRNAs. In that case, it is interesting to know whether a given aminoacyl-tRNA-synthetase is able to recognize any tRNA or only a particular group of tRNAs. The existence of such groups would lead to the concept of tRNA families. For that, we estimated the affinities of non-cognate homologous tRNA species for yeast valyl-tRNA-synthetase by using competition experiments. The measured affinities varied, in standard aminoacylation conditions, between 1:100 to 1:1000 of that of the non-cognate tRNA. In the absence of Mg2+ ions or in the presence of low concentration of this cation, the affinities were higher and could reach 1:3 of the affinity of the cognate tRNA. On the other hand, we determined the inhibitory effect of a high concentration of tRNAVal toward the aminoacylation of tRNAs specific for 13 amino acids. In order to compare the effects, we determined approximate Km/Ki values. These values ranged from 0.07 for methionyl tRNA synthetase to 0.002 for leucyl tRNA synthetase. For some aminoacyl-tRNA-synthetases, the inhibition was too low to be detected by this technique. Two conclusions arise from this study. First, it seems that non-specific recognitions are quite a general phenomenon. Secondly, if one classifies tTNAs according to their affinities for valyl-tRNA-synthetase, it does not appear any well cut group of tRNAs. This result is not conflicting with the fact that on the basis of aminoacylation criteria several authors have found tRNA and aminoacyl-tRNA-synthetase families since we have already shown that discrimination depends rather on the maximal velocity of the reaction than on the affinity between the tRNA and the aminoacyl-tRNA-synthetases. Finally, the non-existence of clear-cut recognition families of tRNAs casts some doubts on the approach consisting in the characterisation of recognition sites of the tRNAs by the aminoacyl-tRNA-synthetases by comparing the sequences of tRNAs which are amonoacylated by a given aminoacyl-tRNA-synthetase.     

Solvation change and ion release during aminoacylation by aminoacyl-tRNA synthetases

Discrimination between cognate and non-cognate tRNAs by aminoacyl-tRNA synthetases occurs at several steps of the aminoacylation pathway. We have measured changes of solvation and counter-ion distribution at various steps of the aminoacylation pathway of glutamyl- and glutaminyl-tRNA synthetases. The decrease in the association constant with increasing KCl concentration is relatively small for cognate tRNA binding when compared to known DNA–protein interactions. The electro-neutral nature of the tRNA binding domain may be largely responsible for this low ion release stoichiometry, suggesting that a relatively large electrostatic component of the DNA–protein interaction free energy may have evolved for other purposes, such as, target search. Little change in solvation upon tRNA binding is seen. Non-cognate tRNA binding actually increases with increasing KCl concentration indicating that charge repulsion may be a significant component of binding free energy. Thus, electrostatic interactions may have been used to discriminate between cognate and non-cognate tRNAs in the binding step. The catalytic constant of glutaminyl-tRNA synthetase increases with increasing osmotic pressure indicating a water release of 8.4 ± 1.4 mol/mol in the transition state, whereas little change is seen in the case of glutamyl-tRNA synthetase. We propose that the significant amount of water release in the transition state, in the case of glutaminyl-tRNA synthetase, is due to additional contact of the protein with the tRNA in the transition state.     

Influence of various factors on the recognition specificity of tRNAs by yeast valyl-tRNA synthetase.

Using filtration through nitrocellulose membranes we found that complexes between yeast valyl-tRNA synthetase can easily be detected at low pH and ionic strength with the cognate tRNAVal, but also with several non-cognate tRNAs (tRNAPhe, tRNATyr, tRNAMet and tRNAAsp). We show here that the amino acid linked to the tRNA has no detectable effect on these interactions. The influence of various factors on the discrimination by the enzyme between the cognate and the non-cognate tRNAs has been studied. An increase in pH or ionic strength leads to a decrease in the same ratio of the affinity constants between the enzyme and the cognate as well as the noncognate tRNA. The addition of organic solvents has little effect on these constant either in the cognate or in the non-cognate systems; the addition of substrates of the aminoacylation reaction has not effect on the ratio between the constants. This similar behaviour suggests that at least part of the specific of non-specific interactions must be identical. On the contrary, magnesium between 1 mM and 50 mM increases the specificity of recognition, showing the importance of slight conformational changes in the tRNA molecule to the specificity of interaction.     

Effects of adenosine triphosphate and magnesium chloride on affinity elution of aminoacyl-transfer ribonucleic acid synthetases from phosphocellulose with transfer ribonucleic acids.

Aminoacyl-tRNA synthetases of bakers' yeast (Saccharomyces cerevisiae) were adsorbed to a phosphocellulose (P-cellulose) column, and those specific for tyrosine [EC 6.1.1.1], threonine [EC 6.1.1.3], valine [EC 6.1.1.9], and isoleucine [EC 6.1.1.5] were eluted with several specific tRNAs. Elutions of these synthetases were affected by ATP and/or MgCl2. The effects of ATP and MgCl2 differ with synthetases. Elutions of tyrosyl- and valyl-tRNA synthetases with their cognate tRNAs were more specific in the presence of MgCl2. Isoleucyl-tRNA synthetase was eluted with its cognate tRNA in the presence of both ATP and MgCl2. On the other hand, threonyl-tRNA synthetase was eluted in the absence of ATP and MgCl2 with unfractionated tRNA but not with some non-cognate tRNAs. This suggests that elution of threonyl-tRNA synthetase is highly specific. The present data on the effects of ATP or MgCl2 or both on this affinity elution will be useful for simple and rapid purification of the synthetases.     

Protein engineering of homodimeric tyrosyl-tRNA synthetase to produce active heterodimers

Heterodimers of tyrosyl-tRNA synthetase from Bacillus stearothermophilus have been produced by mutagenesis at the subunit interface. Oppositely charged groups have been engineered into the subunits so that they can form a complementary pair. Wild-type tyrosyl-tRNA synthetase is a symmetrical dimer in which the side chains of the 2 Phe-164 residues interact at the subunit interface. Phe-164 was mutated to Asp in tyrosyl-tRNA synthetase and to Lys in a truncated enzyme (des-(321-419)tyrosyl-tRNA synthetase) which lacks the two tRNA-binding sites, but which can catalyze pyrophosphate exchange. The size difference allows subunit association to be studied by gel filtration chromatography. These changes induce reversible dissociation from active dimers into inactive monomers at pH values which favor ionization at position 164. A mixture of the two mutants near neutral pH is apparently fully active in pyrophosphate exchange and consists of a heterodimer of [Asp164]tyrosyl-tRNA synthetase and [Lys164]des-(321-419)tyrosyl-tRNA synthetase. Despite having only one binding site for tRNA, heterodimer has full aminoacylation activity at high concentrations of tyrosine. We have therefore produced a family of dimers that differ in stability near neutral pH. This novel approach using protein engineering allows specific dimerization of subunits of the same size that have different defined mutations, each subunit being tagged by the charge. Such hybrid proteins can be used to study subunit interaction.     

Structure-specific tRNA determinants for editing a mischarged amino acid

Alanyl-tRNA synthetase efficiently aminoacylates tRNAAla and an RNA minihelix that comprises just one domain of the two-domain L-shaped tRNA structure. It also clears mischarged tRNAAla using a specialized domain in its C-terminal half. In contrast to full-length tRNAAla, minihelixAla was robustly mischarged and could not be edited. Addition in trans of the missing anticodon-containing domain did not activate editing of mischarged minihelixAla. To understand these differences between minihelixAla and tRNAAla, several chimeric full tRNAs were constructed. These had the acceptor stem of a non-cognate tRNA replaced with the stem of tRNAAla. The chimeric tRNAs collectively introduced multiple sequence changes in all parts but the acceptor stem. However, although the acceptor stem in isolation (as the minihelix) lacked determinants for editing, alanyl-tRNA synthetase effectively cleared a mischarged amino acid from each chimeric tRNA. Thus, a covalently continuous two-domain structure per se, not sequence, is a major determinant for clearance of errors of aminoacylation by alanyl-tRNA synthetase. Because errors of aminoacylation are known to be deleterious to cell growth, structure-specific determinants constitute a powerful selective pressure to retain the format of the two-domain L-shaped tRNA.     

Regulation of the tyrosine biosynthetic enzymes in Salmonella typhimurium: analysis of the involvement of tyrosyl-transfer ribonucleic acid and tyrosyl-transfer ribonucleic acid synthetase

Mutants of Salmonella typhimurium were isolated that require tyrosine for growth because of an altered tyrosyl-transfer ribonucleic acid (tRNA) synthetase. Extracts of one strain (JK10) contain a labile enzyme with decreased ability to transfer tyrosine to tRNA(Tyr) and a higher K(m) for tyrosine than the wild-type enzyme. Strain JK10 maintains repressed levels of the tyrosine biosynthetic enzymes when the growth rate is restricted due to limitation of charged tRNA(Tyr). Several second-site revertants of strain JK10 exhibit temperature-sensitive growth due to partially repaired, heat-labile tyrosyl-tRNA synthetase. The tyrosine biosynthetic enzymes are not derepressed in thermosensitive strains grown at the restrictive temperature. A class of tyrosine regulatory mutants, designated tyrR, contains normal levels of tyrosyl-tRNA synthetase and tRNA(Tyr). These results suggest that charging of tRNA(Tyr) is not necessary for repression. This conclusion is substantiated by the finding that 4-aminophenylalanine, a tyrosine analogue which causes repression of the tyrosine biosynthetic enzymes, is not attached to tRNA(Tyr) in vivo, nor does it inhibit the attachment reaction in vitro. A combined regulatory effect due to the simultaneous presence of tyrS and tyrR mutations in the same strain was detected. The possibility of direct participation of tyrosyl-tRNA synthetase in tyrosine regulation is discussed.     

Structural elements defining elongation factor Tu mediated suppression of codon ambiguity

In most prokaryotes Asn-tRNA^Asn and Gln-tRNA^Gln are formed by amidation of aspartate and glutamate mischarged onto tRNA^Asn and tRNA^Gln, respectively. Coexistence in the organism of mischarged Asp-tRNA^Asn and Glu-tRNA^Gln and the homologous Asn-tRNA^Asn and Gln-tRNA^Gln does not, however, lead to erroneous incorporation of Asp and Glu into proteins, since EF-Tu discriminates the misacylated tRNAs from the correctly charged ones. This property contrasts with the canonical function of EF-Tu, which is to non-specifically bind the homologous aa-tRNAs, as well as heterologous species formed in vitro by aminoacylation of non-cognate tRNAs. In Thermus thermophilus that forms the Asp-tRNA^Asn intermediate by the indirect pathway of tRNA asparaginylation, EF-Tu must discriminate the mischarged aminoacyl-tRNAs (aa-tRNA). We show that two base pairs in the tRNA T-arm and a single residue in the amino acid binding pocket of EF-Tu promote discrimination of Asp-tRNA^Asn from Asn-tRNA^Asn and Asp-tRNA^Asp by the protein. Our analysis suggests that these structural elements might also contribute to rejection of other mischarged aa-tRNAs formed in vivo that are not involved in peptide elongation. Additionally, these structural features might be involved in maintaining a delicate balance of weak and strong binding affinities between EF-Tu and the amino acid and tRNA moieties of other elongator aa-tRNAs.     

MIST,a Novel Approach to Reveal Hidden Substrate Specificity in Aminoacyl-tRNA Synthetases

Aminoacyl-tRNA synthetases (AARSs) constitute a family of RNA-binding proteins, that participate in the translation of the genetic code, by covalently linking amino acids to appropriate tRNAs. Due to their fundamental importance for cell life, AARSs are likely to be one of the most ancient families of enzymes and have therefore been characterized extensively. Paradoxically, little is known about their capacity to discriminate tRNAs mainly because of the practical challenges that represent precise and systematic tRNA identification. This work describes a new technical and conceptual approach named MIST (Microarray Identification of Shifted tRNAs) designed to study the formation of tRNA/AARS complexes independently from the aminoacylation reaction. MIST combines electrophoretic mobility shift assays with microarray analyses. Although MIST is a non-cellular assay, it fully integrates the notion of tRNA competition. In this study we focus on yeast cytoplasmic Arginyl-tRNA synthetase (yArgRS) and investigate in depth its ability to discriminate cellular tRNAs. We report that yArgRS in submicromolar concentrations binds cognate and non-cognate tRNAs with a wide range of apparent affinities. In particular, we demonstrate that yArgRS binds preferentially to type II tRNAs but does not support their misaminoacylation. Our results reveal important new trends in tRNA/AARS complex formation and potential deep physiological implications.     

Unusual anticodon loop structure found in E.coli lysine tRNA.

Although both tRNA(Lys) and tRNA(Glu) of E. coli possess similar anticodon loop sequences, with the same hypermodified nucleoside 5-methylaminomethyl-2-thiouridine (mnm5s2U) at the first position of their anticodons, the anticodon loop structures of these two tRNAs containing the modified nucleoside appear to be quite different as judged from the following observations. (1) The CD band derived from the mnm5s2U residue is negative for tRNA(Glu), but positive for tRNA(Lys). (2) The mnm5s2U monomer itself and the mnm5s2U-containing anticodon loop fragment of tRNA(Lys) show the same negative CD bands as that of tRNA(Glu). (3) The positive CD band of tRNA(Lys) changes to negative when the temperature is raised. (4) The reactivity of the mnm5s2U residue toward H2O2 is much lower for tRNA(Lys) than for tRNA(Glu). These features suggest that tRNA(Lys) has an unusual anticodon loop structure, in which the mnm5s2U residue takes a different conformation from that of tRNA(Glu); whereas the mnm5s2U base of tRNA(Glu) has no direct bonding with other bases and is accessible to a solvent, that of tRNA(Lys) exists as if in some way buried in its anticodon loop. The limited hydrolysis of both tRNAs by various RNases suggests that some differences exist in the higher order structures of tRNA(Lys) and tRNA(Glu). The influence of the unusual anticodon loop structure observed for tRNA(Lys) on its function in the translational process is also discussed.