Threonine tRNAs and their genes in Escherichia coli

Summary The subject of this study was the threonine isoacceptor family of tRNAs in Escherichia coli and the genes coding for them. The goal was to identify and map all the genes and to determine the relative contribution of each gene to the tRNA pool. The mapping experiments exploited gene-dosage effects in partially diploid strains; if a strain harboring a particular F episome overproduced a particular tRNA species, it could be concluded that the gene for that tRNA was located on the chromosomal segment carried by the F. Isoacceptor tRNAs were distinguished by column fractionation. It was found that there are three major threonine tRNA species that occur in roughly equal amounts. These are tRNA ₁ ^Thr , which is encoded by a gene in the distal region of the rrnD ribosomal RNA operon, and tRNA ₃ ^Thr and tRNA ₄ ^Thr , which come from genes in the cluster thrU tyrU glyT thrT at 89 min on the map. The relative abundances of the tRNA species roughly match the reported frequencies of the codons that they recognize in mRNA. Although the tRNA ₄ ^Thr has a mismatched base pair that raised questions about its biological activity, it was found to be functional at least with respect to recognition by the threonyl-tRNA synthetase. An apparent fourth gene affecting threonine tRNA has been identified and mapped at 6–8 min; it is here designated thrW. It may be a structural gene for a minor tRNA species, present in one-third the amount of each of the major species, and chromatographically indistinguishable from tRNA ₄ ^Thr .A preliminary report of most of this work has appeared previously (M.M. Comer, Abstr. Annu. Meet. Am. Soc. Microbiol. 1980, p. 109)     

Structure and properties of a bovine liver UGA suppressor serine tRNA with a tryptophan anticodon

A bovine liver serine tRNA with a variety of unusual features has been sequenced and characterized. This tRNA is aminoacylated with serine, although it has a tryptophan anticodon CmCA. In ribosome binding assays, this tRNA (tRNA_CmCA^Ser) binds to the termination codon UGA and shows little or no binding in response to a variety of other codons including those for tryptophan and serine. The unusual codon recognition properties of this molecule were confirmed in an in vitro assay where this tRNA suppressed UGA termination. This is the first naturally occurring eucaryotic suppressor tRNA to be so characterized. Other unusual features, possibly related to the ability of this tRNA to read UGA, are the presence of two extra nucleotides, compared to all other tRNAs, between the universal residues U at position 8 and A at position 14 and the presence of an extra unpaired nucleotide within the double-stranded loop IV stem. This tRNA is also the largest eucaryotic tRNA sequenced to date (90 nucleotides). Despite its size, however, it contains only six modified residues. tRNA_CmCA^Ser shows extremely low homology to other mammalian serine (47–52% homology) or tryptophan (49% homology) tRNAs.     

Localization of tRNA genes of Drosophila melanogaster by in situ hybridization

Six purified tRNAs labeled with ¹²⁵I by chemical or enzymatic methods were hybridized to polytene chromosomes of Drosophila melanogaster. The main chromosomal regions of hybridization were: tRNA _GGA ^Gly , 58A, 84C, and 90E; tRNA ₂ ^Leu , 44E, 66B5-8, and 79F; tRNA _2b ^Ser , 86A, 88A9-12, and 94A6-8; tRNA ₃ ^Thr , 47F and 87B; tRNA ₄ ^Thr , 93A1-2; and tRNA ₁ ^Tyr , 19F, 22F-23A, 41, 50C1-4 and 85A. At 50C the hybridization of tRNA ₁ ^Tyr was polymorphic in the giant strains. When the hybridization of three valine isoacceptors studied previously was re-investigated, it was found that only one hybridization site, 90BC, was shared between tRNA _3b ^Val and tRNA ₄ ^Val . tRNA _3a ^Val did not have any sites in common with the other two.     

Studies on the ribothymidine content of specific rat and human tRNAs: a postulated role for 5-methyl cytosine in the regulation of ribothymidine biosynthesis.

Total mammalian tRNAs contain on the average less than one mole of ribothymidine per mole of tRNA. Mammalian tRNAs can be grouped into at least four classes, depending upon their ribothymidine content at position 23 from the 3′ terminus. Class A contains tRNA in which a nucleoside other than uridine replaces ribothymidine (tRNA_i^Met); Class B contains tRNA in which one mole of a modified uridine (rT, ψ, or 2′-O-methylribothymidine) is found per mole of tRNA (tRNA^Ser, tRNA^Trp, and tRNA^Lys, respectively). Class C contains tRNA in which there is a partial conversion of uridine to ribothymidine (tRNA^Phe, tRNA₁^Gly, tRNA₂^Gly); Class D contains tRNA which totally lacks ribothymidine (tRNA^Val). Only those tRNAs in Class C are acceptable substrates for $\text{E.}\text{coli}$ uridine methylase, under the conditions used in these studies. These observations cannot be adequately explained solely on the basis of the presence or absence of a specific “universal” nucleoside other than U or rT at position 23 from the 3′ terminus. However, correlations can be made between the ribothymidine and 5-methylcytosine content of eucaryotic tRNA. We postulate that the presence of one or more 5-methylcytosines in and adjacent to loop III (minor loop) in individual tRNAs act to regulate the amount of ribothymidine formed by uridine methylase. Several experiments are proposed as tests for this hypothesis.     

Functional importance of Ψ38 and Ψ39 in distinct tRNAs,amplified for tRNAGln(UUG) by unexpected temperature sensitivity of the s2U modification in yeast

The numerous modifications of tRNA play central roles in controlling tRNA structure and translation. Modifications in and around the anticodon loop often have critical roles in decoding mRNA and in maintaining its reading frame. Residues U₃₈ and U₃₉ in the anticodon stem–loop are frequently modified to pseudouridine (Ψ) by members of the widely conserved TruA/Pus3 family of pseudouridylases. We investigate here the cause of the temperature sensitivity of pus3Δ mutants of the yeast Saccharomyces cerevisiae and find that, although Ψ₃₈ or Ψ₃₉ is found on at least 19 characterized cytoplasmic tRNA species, the temperature sensitivity is primarily due to poor function of tRNA^Gln(UUG), which normally has Ψ₃₈. Further investigation reveals that at elevated temperatures there are substantially reduced levels of the s²U moiety of mcm⁵s²U₃₄ of tRNA^Gln(UUG) and the other two cytoplasmic species with mcm⁵s²U₃₄, that the reduced s²U levels occur in the parent strain BY4741 and in the widely used strain W303, and that reduced levels of the s²U moiety are detectable in BY4741 at temperatures as low as 33°C. Additional examination of the role of Ψ_38,39 provides evidence that Ψ₃₈ is important for function of tRNA^Gln(UUG) at permissive temperature, and indicates that Ψ₃₉ is important for the function of tRNA^Trp(CCA) in trm10Δ pus3Δ mutants and of tRNA^Leu(CAA) as a UAG nonsense suppressor. These results provide evidence for important roles of both Ψ₃₈ and Ψ₃₉ in specific tRNAs, and establish that modification of the wobble position is subject to change under relatively mild growth conditions.     

Unexpected expansion of tRNA substrate recognition by the yeast m1G9 methyltransferase Trm10

N-1 Methylation of the nearly invariant purine residue found at position 9 of tRNA is a nucleotide modification found in multiple tRNA species throughout Eukarya and Archaea. First discovered in Saccharomyces cerevisiae, the tRNA methyltransferase Trm10 is a highly conserved protein both necessary and sufficient to catalyze all known instances of m¹G₉ modification in yeast. Although there are 19 unique tRNA species that contain a G at position 9 in yeast, and whose fully modified sequence is known, only 9 of these tRNA species are modified with m¹G₉ in wild-type cells. The elements that allow Trm10 to distinguish between structurally similar tRNA species are not known, and sequences that are shared between all substrate or all nonsubstrate tRNAs have not been identified. Here, we demonstrate that the in vitro methylation activity of yeast Trm10 is not sufficient to explain the observed pattern of modification in vivo, as additional tRNA species are substrates for Trm10 m¹G₉ methyltransferase activity. Similarly, overexpression of Trm10 in yeast yields m¹G₉ containing tRNA species that are ordinarily unmodified in vivo. Thus, yeast Trm10 has a significantly broader tRNA substrate specificity than is suggested by the observed pattern of modification in wild-type yeast. These results may shed light onto the suggested involvement of Trm10 in other pathways in other organisms, particularly in higher eukaryotes that contain up to three different genes with sequence similarity to the single TRM10 gene in yeast, and where these other enzymes have been implicated in pathways beyond tRNA processing.     

Rabbit liver tRNAVal1: II. Unusual secondary structure of T ψ C stem and loop due to a U54:A60 base pair

In contrast to all other known tRNAs, mammalian tRNA^Val₁ contains two adenosines A₅₉ and A₆₀, opposite to U₅₄ and ψ₅₅ in the UψCG sequence of the TψC loop, which could form unusual A:U (or A:ψ) pairs in addition to the five “normal” G:C pairs. In order to measure the number of G:C and A:U (A:ψ) pairs in the TψC stem, we prepared the 30 nucleotide long 3′-terminal fragment of this tRNA by “m⁷G-cleavage”. From differentiated melting curves and temperature jump experiments it was concluded that the TψC stem in this fragment is in fact extended by an additional A₆₀:U₅₄ pair. A dimer of this fragment with 14 base pairs was characterized by gel electrophoresis and by the same physical methods. An additional A:U pair in the tRNA^Val₁ fragment does not necessarily mean that this is also true for intact tRNA. However, we showed that U₅₄ is far less available for enzymatic methylation in mammalian tRNA^Val₁ compared to tRNA from T⁻E. coli. This clear difference in U₅₄ reactivity, together with the identification of an extra A₆₀:U₅₄ pair in the UψCG containing fragment suggests the presence of a 6 base pair TψC stem and a 5 nucleotide TψC loop in this tRNA.     

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.     

tRNA2Lys gene clusters in Drosophila

We have isolated segments of Drosophila melanogaster DNA that contain two clusters of tRNA₂^Lys genes. In one segment, pPW511, there is a cluster of three of these genes surrounded by other tRNA genes. Two other segments, pPW516 and pPW541. share a 3 × 10³ base-pair region that has a cluster of four tRNA₂^Lys genes. This cluster is flanked by 20 × 10³ base-pairs of DNA that does not appear to have other tRNA genes. The tRNA genes in both clusters are irregularly spaced and are intermingled with moderately repetitive DNA. Each cluster is present once or perhaps twice in the haploid genome and has the same arrangement of restriction endonuclease sites in the genomic DNA as in the isolated, cloned DNA. In situ hybridization to polytene chromosomes localized the pPW511 cluster to the 42A region and the pPW516/541 cluster to the 42E region. Another region, 50B, also contains tRNA₂^Lys genes. In sum, these cloned tRNA₂^Lys genes account for most of this gene family and are irregularly spaced in two clusters.     

Secondary structure in transfer RNA genes

The bacterial strand of the heteroduplex of λh80 dglyTsu⁺₃₆tyrTthrT with λh80 carries a cluster of three transfer RNA genes. The bacterial strands of the heteroduplexes of φ80hpsu^+,?_III and φ80hpsu^?_III with φ80h carry two and one genes for tyrosine tRNA, respectively. When these heteroduplexes are spread under weakly denaturing conditions (low formamide), secondary structure features consisting of one or several closely clustered, short duplex regions (folds) are observed. The features map at the positions of the tRNA gene clusters. They are not seen if the DNA is hybridized to Escherichia coli tRNA. It is concluded that the secondary structure features are due to self-complementary sequences in the tRNA genes. In some cases, the duplex folds appear to involve base pairing between sequences on different tRNA genes of a cluster and may also involve the spacer sequences between the tRNA sequences.     

Nucleotide sequences of yeast genes for tRNA(2), tRNA(2) and tRNA(1): homology blocks occur in the vicinity of different tRNA genes

Three members of a collection of pBR322-yeast DNA recombinant plasmids containing yeast tRNA genes have been analyzed and sequenced. Each plasmid carries a single tRNA gene: pY44, tRNA^Ser₂; pY41, tRNA^Arg₂; pY7, tRNA^Val₁. All three genes are intronless and terminate in a cluster of Ts in the non-coding strand. The sequence information here and previously determined sequences allow an extensive comparison of the regions flanking several yeast tRNA genes. This analysis has revealed novel features in tRNA gene arrangement. Blocks of homology in the flanking regions were found between the tRNA genes of an isoacceptor family but, more interestingly, also between genes coding for tRNAs of different amino-acid specificities. Particularly, three examples are discussed in which sequence elements in the neighborhood of different tRNA genes have been conserved to a high degree and over long distances.     

Purification Of Methionine Acceptor tRNA On Arginine-Agarose

Crude E. coli tRNA or enriched methionine acceptor tRNA can be separated into three stiecies on a column of arginine-agarose. The first peak eluted is tRNA^Met and the latter two peaks are two forms of tRNA^Met _f. From crude tRNA, tRNA^Met _m is obtained in approximately 50% purity. Arginine-agarose separates enriched methionine accepting tRNA into three homogeneous fractions.     

Modified constructs of the tRNA TPsiC domain to probe substrate conformational requirements of m(1)A(58) and m(5)U(54) tRNA methyltransferases

The TΨC stem and loop (TSL) of tRNA contains highly conserved nucleoside modifications, m⁵C₄₉, T₅₄, Ψ₅₅ and m¹A₅₈. U₅₄ is methylated to m⁵U (T) by m⁵U₅₄ methyltransferase (RUMT); A₅₈ is methylated to m¹A by m¹A₅₈ tRNA methyltransferase (RAMT). RUMT recognizes and methylates a minimal TSL heptadecamer and RAMT has previously been reported to recognize and methylate the 3′-half of the tRNA molecule. We report that RAMT can recognize and methylate a TSL heptadecamer. To better understand the sensitivity of RAMT and RUMT to TSL conformation, we have designed and synthesized variously modified TSL constructs with altered local conformations and stabilities. TSLs were synthesized with natural modifications (T₅₄ and Ψ₅₅), naturally occurring modifications at unnatural positions (m⁵C₆₀), altered sugar puckers (dU₅₄ and/or dU₅₅) or with disrupted U-turn interactions (m¹Ψ₅₅ or m¹m³Ψ₅₅). The unmodified heptadecamer TSL was a substrate of both RAMT and RUMT. The presence of T₅₄ increased thermal stability of the TSL and dramatically reduced RAMT activity toward the substrate. Local conformation around U₅₄ was found to be an important determinant for the activities of both RAMT and RUMT.     

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.     

Codon binding and translational properties of an isoaccepting lysine tRNA peculiar to virus-transformed Cells

Summary Isoaccepting lysyl-tRNAs from virus-transformed cells in culture were fractionated in the RPC-5 system into peaks 1, 2, 4, 5a, 5, and 6. tRNA ₆ ^Lys previously was found predominantly associated with transformed cells. The codon response of each peak was determined in an E. coli ribosomal binding assay. tRNA ₁ ^Lys , tRNA ₂ ^Lys , and tRNA ₄ ^Lys are highly specific for the ⁵AAG³ codon. tRNA ₅ ^Lys and tRNA _5a ^Lys preferentially bind in response to AAA. tRNA ₆ ^Lys binds in response to AAA 3-fold better than in response to AAG. The presence of thiolated nucleosides in the anticodon regions of tRNA _5a ^Lys , tRNA ₅ ^Lys , and tRNA ₆ ^Lys is indicated by I₂-inactivation of aminoacylation ability with no effect on the other isoacceptors.Functional abilities of the isoacceptors were compared in a wheat germ translational system with tobacco mosaic virus RNA as messenger. All of the isoacceptors function about equally well in translation except for tRNA ₆ ^Lys , which is only 14 to 24% as effective as the other isoacceptors.     

Affinity labelling of tryptophanyl-transfer RNA synthetase

A double affinity-labelling approach has been developed in order to convert an oligomeric enzyme with multiple active centres into a single-site enzyme.Tryptophanyl-transfer RNA synthetase (EC 6.1.1.2) from beef pancreas is a symmetric dimer, α₂ An ATP analogue, γ-(p-azidoanilide)-ATP does not serve as a substrate for enzymatic aminoacylation of tRNA^Trp but acts as an effective competitive inhibitor in the absence of photochemical reaction, with K₁ = 1 × 10^?3m (K_mfor ATP = 2 × 10^?4m). The covalent photoaddition of azido-ATP³ results in complete loss of enzymatic activity in both the ATP-[³²P]pyrophosphate exchange reaction and tRNA aminoacylation. ATP completely protects the enzyme against inactivation. However, covalent binding of azido-ATP is also observed outside the active centres. The difference between covalent binding of the azido-ATP in the absence and presence of ATP corresponds to 2 moles of the ATP analogue per mole of the enzyme.Two binding sites for tRNA^Trp have been found from complex formation at pH 5.8 in the presence of Mg²⁺. The two tRNA molecules bind, with K_dis = 3.6 × 10^?8m and K_dis = 0.9 × 10^?6m, respectively, pointing to a strong negative co-operativity between the binding sites for tRNA.N-chlorambucilyl-tryptophanyl-tRNA^Trp and TRSase form a complex with K_dis = 5.5 × 10^?8m at pH 5.8 in the presence of 10 mm-Mg²⁺. This value is similar to the value of K_dis for tryptophanyl-tRNA of 4.8 × 10^?8m. Under the same conditions a 1:1 complex (in mol) is formed between the enzyme and Trp-tRNA or N-chlorambucilyl-Trp-tRNA. On incubation, a covalent bond is formed between N-chlorambucilyl-Trp-tRNA and TRSase; 1 mole of affinity reagent alkylates 1 mole of enzyme independently of the concentration of the modifier. The alkylation reaction is completely inhibited by the presence of tRNA^Trp whereas the tRNA devoid of tRNA^Trp does not affect the rate of alkylation. In the presence of either ATP or tryptophan, or a mixture of the two, the alkylation reaction is inhibited even though these ligands have no effect on the complex formation between TRSase and the tRNA analogue. Photoaddition of the azido-ATP completely prevents the reaction of the enzyme with the tRNA analogue, although the non-covalent complex formation is not affected.Exhaustive alkylation of TRSase partially inhibits the reaction of ATP [³²P]pyrophosphate exchange and completely blocks the aminoacylation of tRNA^Trp. Cleavage of the tRNA which is covalently bound to TRSase restores both the ATP-[³²P]pyrophosphate exchange and aminoacylation activity.The TRSase which is covalently-bound to R-Trp-tRNA is able to incorporate only one ATP molecule per dimeric enzyme into the active centre. This doubly modified enzyme is completely enzymatically inactive. Removal of the tRNA residue from the doubly modified enzyme results in the formation of the derivative with one blocked ATP site. Therefore, a “single-site” TRSase may be generated either by alkylation of the enzyme with Cl-R-Trp-tRNA or after the removal of covalently bound tRNA from the doubly labelled protein.Tryptophanyl-tRNA synthetase containing blocked ATP and/or tRNA binding site(s) seems to bo a useful tool for investigation of negative co-operativity and may help in the elucidation of the structure function relationships between the active centres.     

The developmental pattern of homologous and heterologous tRNA methylation in rat brain differential effect of spermidine

UsingS-adenosyl-L-[Me-¹⁴C] methionine, rat cerebral cortex methyltransferase activity was determined during the early postnatal period in the absence of addedEscherichia coli tRNA and in its presence. [Me-¹⁴C] tRNA was purified from both systems and its [Me-¹⁴C] base composition determined. The endogenous formation of [Me-¹⁴C] tRNA (homologous tRNA methylation) was totally abolished in the presence of 2.5 mM spermidine, whereasE. coli B tRNA methylation (heterologous methylation) was markedly stimulated. Only [Me-¹⁴C] 1-methyl guanine and [Me-¹⁴C]N ²-methyl guanine were formed by homologous methylation, there being an inverse shift in their relative proportions with age. Heterologous tRNA methylation led, additionally, to the formation of [Me-¹⁴C]N ₂ ² -dimethyl guanine, 5-methyl cytosine, 1-methyl adenine, 5-methyl uracil, 2-methyl adenine, and 1-methyl hypoxanthine. A comparison of heterologous tRNA methylation between the whole brain cortex (containing nerve and glial cells) and bulk-isolated nerve cell bodies revealed markedly lower proportions of [Me-¹⁴C]N ₂-methyl andN ₂ ² -dimethyl guanine and significantly higher proportions of [Me-¹⁴C] 1-methyl adenine in the neurons. The present findings suggest (1) that homologous tRNA methylation may provide developing brain cells with continuously changing populations of tRNA and (2) that neurons are enriched in adenine residue-specific tRNA methyltransferases that are highly sensitive to spermidine.This research was supported by grant NS-06294 of the United States Public Health Service.     

Selective modification of cytidine, uridine, guanosine and pseudouridine residues in Escherichia coli leucine transfer ribonucleic acid

The specificity of methoxyamine for the cytidine residues in an Escherichia coli leuoine transfer RNA (tRNA₁^leu is described in detail. Of the six non-hydrogen-bonded cytidine residues in the clover-leaf model of this tRNA, four are very reactive (C-35, 53, 85 and 86) and two are unreactive (C-67 and 79).The specificity of l-cyclohexyl-3-[2-morpholino-(4)-ethyl]carbodiimide methotosylate for the uridine, guanosine and pseudouridine residues in the leucine tRNA was also investigated. The carbodiimide completely modified four uridine residues (U-33, 34, 50 and 51) and partially modified G-37 and Ψ-39. For technical reasons, the sites of partial modification in loop I of the tRNA were difficult to establish. There was no modification of base residues in loop IV nor of U-59 at the base of stem e of the tRNA.The modification patterns described for the leucine tRNA are compared with those observed for the E. coli initiator tRNA₁^met and su⁺_III tyrosine tRNA. Several general conclusions regarding tRNA conformation are made. In particular, the evidence supporting a diversity of anticodon loop structures amongst tRNAs is discussed.     

Identification of the Balbiani ring 2 chromomere and determination of the content and compaction of its DNA

Highly purified tRNAs from Drosophila melanogaster were iodinated with ¹²⁵I and hybridized to squashes of polytene chromosomes of Drosophila salivary glands followed by autoradiography to localize binding sites. Most tRNAs hybridize strongly to more than one site and weakly to one or more additional sites. The major sites for various tRNAs are the following: tRNA ₂ ^Arg , 42A, 84F_1,2; tRNA ₂ ^Asp , 29DE; tRNA ₃ ^Gly , 22BC, 35BC, 57BC; tRNA ₂ ^Lys , 42A, 42E; tRNA ₅ ^Lys , 84AB, 87B; tRNA ₂ ^Met , 48B_5–7, 72F_1–2, 83F-84A; tRNA ₃ ^Met , 46A_1–2, 61D_1–2, 70F_1–2; tRNA ₄ ^Ser , 12DE, 23E; tRNA ₇ ^Ser , 12DE, 23E; tRNA _3a ^Val , 64D; tRNA _3b ^Val , 84D_3–4, 92B_1–9; tRNA ₄ ^Val , 56D_3–7, 70BC.