Nucleotide sequence of three isoaccepting lysine tRNAs from rabbit liver and SV40-transformed mouse fibroblasts.

The lysine isoacceptor tRNAs differ in two aspects from the majority of the other mammalian tRNA species: they do not contain ribosylthymine (T) in loop IV, and a 'new' lysine tRNA, which is practically absent in non-dividing tissue, appears at elevated levels in proliferating cells. We have therefore purified the three major isoaccepting lysine tRNAs from rabbit liver and the 'new' lysine tRNA isolated from SV40-transformed mouse fibroblasts, and determined their nucleotide sequences. Our basic findings are as follows. a) The three major lysine tRNAs (species 1, 2 and 3) from rabbit liver contain 2'-O-methylribosylthymine (Tm) in place of T. tRNA1Lys and tRNA2Lys differ only by a single base pair in the middle of the anticodon stem; the anticodon sequence C-U-U is followed by N-threonyl-adenosine (t6A). TRNA3Lys has the anticodon S-U-U and contains two highly modified thionucleosides, S (shown to be 2-thio-5-carboxymethyl-uridine methyl ester) and a further modified derivative of t6 A (2-methyl-thio-N6-threonyl-adenosine) on the 3' side of the anticodon. tRNA3Lys differs in 14 and 16 positions, respectively, from the other two isoacceptors. b) Protein synthesis in vitro, using synthetic polynucleotides of defined sequence, showed that tRNA2Lys with anticodon C-U-U recognized A-A-G only, whereas tRNA3Lys, which contains thio-nucleotides in and next to the anticodon, decodes both lysine codons A-A-G and A-A-A, but with a preference for A-A-A. In a globin-mRNA-translating cell-free system from ascites cells, both lysine tRNAs donated lysine into globin. The rate and extent of lysine incorporation, however, was higher with tRNA2Lys than with tRNA3Lys, in agreement with the fact that alpha-globin and beta-globin mRNAs contain more A-A-G than A-A-A- codons for lysine. c) A comparison of the nucleotide sequences of lysine tRNA species 1, 2 and 3 from rabbit liver, with that of the 'new' tRNA4Lys from transformed and rapidly dividing cells showed that this tRNA is not the product of a new gene or group of genes, but is an undermodified tRNA derived exclusively from tRNA2Lys. Of the two dihydrouridines present in tRNA2Lys, one is found as U in tRNA4Lys; the purine next to the anticodon is as yet unidentified but is known not be t6 A. In addition we have found U, T and psi besides Tm as the first nucleoside in loop IV.     

Tumor-specific, hypomodified phenylalanyl-tRNA is utilized in translation in preference to the fully modified isoacceptor of normal cells

Phenylalanine transfer RNA (tRNAPhe) of mammalian tissues contains the hypermodified guanine derivative Y (Wye) adjacent to the 3'-end of the anticodon and two O-methylated bases in the 5' portion of the anticodon loop. These positions are hypomodified in a variety of tumor cells including a mouse neuroblastoma. The normal and tumor-specific Phe-tRNAPhe iso-acceptors were prepared from mouse liver and mouse neuroblastoma cells and compared for their activity in incorporating phenylalanine into each phenylalanine site of rabbit globin in a reticulocyte cell-free protein synthesizing system. The hypomodified Phe-tRNAPhe of neuroblastoma cells is generally preferred to the fully modified tRNAPhe of liver in globin synthesis by about 15%. This preference is the same in the translation of both phenylalanine codons, UUC and UUU, but the ratios of incorporation by the Phe-tRNAPhe species vary from site to site within a 2-fold range. Only 2 of 16 phenylalanine residues are donated preferentially by the fully modified Phe-tRNAPhe. One such residue occurs in beta-42, the second of two tandem phenylalanine residues (both encoded by UUC), while the hypomodified isoacceptor is preferred in translation of the first residue. This result indicates that the translation of tandem residues is particularly affected by the tRNAs available. Since the tumor-specific hypomodified Phe-tRNAPhe is generally utilized preferntially, it appears that the bulky Y base and/or other modifications of normal tRNAPhe may modulate protein synthesis and that tumor cells may achieve a growth advantage if their tRNAPhe is hypomodified.     

The effect of the Q base modification on the usage of tRNAHis in globin synthesis

The incorporation of histidine by two competing histidine isoaccepting tRNA species into rabbit globin in a rabbit reticulocyte lysate was studied. The results show that incorporation by each isoacceptor is in proportion to its abundance, indicating that neither species is used preferentially. In a previous study (McNamara and Smith (1978) J. Biol. Chem. $\text{253}$, 5964–5970) we showed that neither tRNA^His species responds preferentially to either of the histidine codons and that there is no preferential incorporation by either species into any histidine-containing site in either globin subunit. The Q base modification is found in one tRNA^His isoacceptor while the other is hypomodified in this this characteristic. The results indicate that none of the aspects of tRNA function in translation that have been examined is affected by Q base.     

Selenium-containing tRNA(Glu) and tRNA(Lys) from Escherichia coli: purification, codon specificity and translational activity

In response to low (approximately 1 microM) levels of selenium, Escherichia coli synthesizes tRNA(Glu) and tRNA(Lys) species that contain 5-methylaminomethyl-2-selenouridine (mnm5Se2U) instead of 5-methylaminomethyl-2-thiouridine (mnm5S2U). Purified glutamate- and lysine-accepting tRNAs containing either mnm5Se2U (tRNA(SeGlu), tRNA(SeLys] or mnm5S2U (tRNA(SGlu), tRNA(SLys] were prepared by RPC-5 reversed-phase chromatography, affinity chromatography using anti-AMP antibodies and DEAE-5PW ion-exchange HPLC. Since mnm5Se2U, like mnm5S2U, appears to occupy the wobble position of the anticodon, the recognition of glutamate codons (GAA and GAG) and lysine codons (AAA and AAG) was studied. While tRNA(SGlu) greatly preferred GAA over GAG, tRNA(SeGlu) showed less preference. Similarly, tRNA(SGlu) preferred AAA over AAG, while tRNA(SeLys) did not. In a wheat germ extract--rabbit globin mRNA translation system, incorporation of lysine and glutamate into protein was generally greater when added as aminoacylated tRNA(Se) than as aminoacylated tRNA(S). In globin mRNA the glutamate and lysine codons GAG and AAG are more numerous than GAA and AAA, thus a more efficient translation of globin message with tRNA(Se) might be expected because of facilitated recognition of codons ending in G.     

Improvement of reading frame maintenance is a common function for several tRNA modifications

Transfer RNAs from all organisms contain many modified nucleosides. Their vastly different chemical structures, their presence in different tRNAs, their occurrence in different locations in tRNA and their influence on different reactions in which tRNA participates suggest that each modified nucleoside may have its own specific function. However, since the frequency of frameshifting in several different mutants [mnmA, mnmE, tgt, truA (hisT), trmD, miaA, miaB and miaE] defective in tRNA modification was higher compared with the corresponding wild-type controls, these modifications have a common function: they all improve reading frame maintenance. Frameshifting occurs by peptidyl-tRNA slippage, which is influenced by the hypomodified tRNA in two ways: (i) a hypomodified tRNA in the ternary complex may decrease the rate by which the complex is recruited to the A-site and thereby increasing peptidyl-tRNA slippage; or (ii) a hypomodified peptidyl-tRNA may be more prone to slip than its fully modified counterpart. We propose that the improvement of reading frame maintenance has been and is the major selective factor for the emergence of new modified nucleosides.     

The effects of a post-transcriptional modification on the function of tRNALys isoaccepting species in translation

Isoacceptors of rabbit liver tRNALys which preferentially translate the codon AAG were compared for their function in several aspects of translation. As shown in other laboratories, Lys-tRNALys1,2 are two isoacceptors which differ from each other by a single base pair and are fully modified with N6-threonyl-adenosine adjacent to the anticodon. Lys-tRNALys4, which occurs commonly in rapidly dividing mammalian cells and tissues, is hypomodified at several bases and contains a precursor of N6-threonyl-adenosine next to its anticodon. These isoacceptors were incubated in cell-free protein synthesizing systems which contain rabbit globin mRNA. (Lys-tRNALys3 which translates AAA was also included.) The resulting globin was isolated and digested with trypsin, and the relative incorporation of lysine from Lys-tRNALys1,2 and from Lys-tRNALys4 into lysine-containing sites in the globin peptides as determined. Lys-tRNALys1,2 and Lys-tRNALys4 translate AAG preferentially, but Lys-tRNALys4 wobbles more than the former and translates AAA codons more efficiently. Overall, Lys-tRNALys1,2 is preferred in globin synthesis by about 30% compared to Lys-tRNALys4, and with one exception, the incorporation of lysine into the individual AAG lysine-containing sites in globin occurs more efficiently from Lys-tRNALys1,2. There is, however, considerable variation from site to site in the relative efficiencies of the Lys-tRNAs in incorporation.     

Alterations in lysine transfer RNA during erythroid differentiation of the Friend cell.

The proportion of lysine tRNA represented by the isoacceptor species lysine tRNA4 has previously been shown to be largest in cells with the greatest ability to proliferate. Using reverse phase chromatography (RPC-5), we have analyzed the changes in the relative quantities of lysine tRNA species which occur in different cellular states of the Friend cell, a transformed murine cell infected with Friend erythroleukemia virus complex. This cell undergoes erythroid differentiation when exposed to various chemicals. Lysine tRNA4 comprises 32% of the total lysine tRNA in rapidly dividing, uninduced Friend cells, but only 16% of the total lysine tRNA in uninducase. Friend cells undergoing erythroid differentiation divide more slowly than uninduced cells, and finally cease proliferation, but lysine tRNA4 becomes the major lysine tRNA species (greater than 50%). This does not appear to reflect erythroid properties of the cell, since the lysine tRNA of the mouse reticulocyte contains very little lysine tRNA4. The non-dividing erythroid Friend cell, therefore, represents an exception to the finding that non-dividing cells usually have little or no lysine tRNA4 present.     

Modified nucleotides in Bacillus subtilis tRNA(Trp) hyperexpressed in Escherichia coli.

In the present study, modified nucleotides in the B. subtilis tRNA(Trp) cloned and hyperexpressed in E. coli have been identified by TLC and HPLC analyses. The modification patterns of the two isoacceptors of cloned B. subtilis tRNA(Trp) have been compared with those of native tRNA(Trp) from B. subtilis and from E. coli. The modifications of the A73 mutant of B. subtilis tRNA(Trp), which is inactive toward its cognate TrpRS, were also investigated. The results indicate the formation of the modified nucleotides S4U8, Gm18, D20, Cm32, i6A/ms2i6A37, T54 and psi 55 on cloned B. subtilis tRNA(Trp). This modification pattern resembles the pattern of E. coli tRNA(Trp), except that m7G is missing from the cloned tRNA(Trp), probably on account of its short extra loop. In contrast, the pattern departs substantially from that of native B. subtilis tRNA(Trp). Therefore, the cloned B. subtilis tRNA(Trp) has taken on largely the modification pattern of E. coli tRNA(Trp) despite the 26% sequence difference between the two species of tRNA, gaining in particular the Cm32 and Gm18 modifications from the E. coli host. A notable difference between the isoacceptors of the cloned tRNA(Trp) was seen in the extent of modification of A37, which occurred as either the hypomodified i6A or the hypermodified ms2i6A form. Surprisingly, base substitution of guanosine by adenosine at position 73 of the cloned tRNA(Trp) has led to the abolition of the 2'-O-methylation modification of the remote G18 residue.     

The major RNA in prosomes of HeLa cells and duck erythroblasts is tRNA(Lys,3).

Two-dimensional gel electrophoresis of HeLa cell prosomal RNAs, 3'-end labeled by RNA ligase, revealed one prominent spot. Determination of a partial sequence at the 3'-end indicated full homology to the 18 nucleotides at the 3'-end of tRNA(Lys,3) from rabbit, the bovine and the human species. An oligonucleotide complementary to the 3'-end of tRNA(Lys,3) hybridized on Northern blots with prosomal RNA from both HeLa cells and duck erythroblasts. In two-dimensional PAGE, the major pRNA of HeLa cells co-migrated with bovine tRNA(Lys,3). Reconstitution of the CCA 3'-end of RNA from both human and duck prosomes, by tRNA-nucleotidyl-transferase, confirmed the tRNA character of this type of RNA. Furthermore, it revealed at least one additional tRNA band about 85 nt long among the prosomal RNA from both species. Finally, confirming an original property of prosomal RNA, we show that in vitro synthesized tRNA(Lys,3) hybridizes stably to duck globin mRNA, and to poly(A)(+)- and poly(A)(-)-RNA from HeLa cells.     

Isoaccepting lysine transfer ribonucleic acid species of Pseudomonas aeruginosa.

Pseudomonas aeruginosa tRNA was treated with iodine, CNBr and N-ethylmaleimide, three thionucleotide-specific reagents. Reaction with iodine resulted in extensive loss of acceptor activity by lysine tRNA, glutamic acid tRNA, glutamine tRNA, serine tRNA and tyrosine tRNA. CNBr treatment resulted in high loss of acceptor ability by lysine tRNA, glutamic acid tRNA and glutamine tRNA. Only the acceptor ability of tyrosine tRNA was inhibited up to 66% by N-ethylmaleimide treatment, a reagent specific for 4-thiouridine. By the combined use of benzoylated DEAE-cellulose and DEAE-Sephadex columns, lysine tRNA of Ps. aeruginosa was resolved into two isoaccepting species, a major, tRNA Lys1 and a minor, tRNALys1. Co-chromatography of 14C-labelled tRNALys1 and 3H-labelled tRNALys2 on benzoylated DEAE-cellulose at pH 4.5 gave two distinct, non-superimposable profiles for the two activity peaks, suggesting that they were separate species. The acceptor activity of these two species was inhibited by about 95% by iodine and CNBr. Both the species showed equal response to codons AAA and AAG and also for poly(A) and poly(A1,G1) suggesting that the anticodon of these species was UUU. Chemical modification of these two species by iodine did not inhibit the coding response. The two species of lysine of Ps. aeruginosa are truly redundant in that they are indistinguishable either by chemical modification or by their coding response.     

A preferential role for lysyl-tRNA4 in the synthesis of diadenosine 5',5'-P1,P4-tetraphosphate by an arginyl-tRNA synthetase-lysyl-tRNA synthetase complex from rat liver

The synthesis of diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) can be catalyzed in vitro by a tetrameric tRNA synthetase complex from rat liver containing two lysyl-tRNA synthetase and two arginyl-tRNA synthetase subunits. This reaction required ATP, AMP, 50-100 microM zinc, and inorganic pyrophosphatase. We show here that AMP can be omitted from the reaction and that the zinc levels can be markedly reduced provided catalytic amounts of tRNA(Lys) are added to the reaction mixture. Ap4A synthesis with purified tRNA(Lys) isoacceptors showed that the minor species, tRNA(4Lys), was 3-fold more active than either of the two major tRNA(Lys) species, tRNA(2Lys) and tRNA(5Lys). No activity could be demonstrated with tRNA(Lys) from Escherichia coli or with tRNA(Lys) or tRNA(Phe) from yeast. Aminoacylation of tRNA(4Lys) was strictly required as determined by the fact that Ap4A synthesis was not observed until aminoacylation was nearly complete, inhibitors of aminoacylation blocked Ap4A synthesis, and there was a strict requirement for added lysine. None of the above observations could be demonstrated, however, when lysyl-tRNA(Lys) was directly supplied to the reaction mixture. Optimum Ap4A synthesis was obtained by the addition of 1 mol of tRNA(Lys)/mol of the synthetase complex. This reaction is unique because it does not require the prior formation of an aminoacyl-AMP intermediate and because it can actively synthesize Ap4A at physiological zinc concentrations. The preferential role for tRNA(4Lys) in Ap4A synthesis is consistent with its prior implication in cell division.     

Nucleotide sequence of a lysine tRNA from Bacillus subtilis.

A lysine tRNA (tRNA1Lys) was purified from Bacillus subtilis W168 by a consecutive use of several column chromatographic systems. The nucleotide sequence was determined to be pG-A-G-C-C-A-U-U-A-G-C-U-C-A-G-U-D-G-G-D-A-G-A-G-C-A-U-C-U-G-A-C-U-U(U*)-U-U-K-A-psi-C-A-G-A-G-G-m7G(G)-U-C-G-A-A-G-G-T-psi-C-G-A-G-U-C-C-U-U-C-A-U-G-G-C-U-C-A-C-C-AOH, where K and U* are unidentified nucleosides. The nucleosides of U34 and m7G46 were partially substituted with U* and G, respectively. The binding ability of lysyl-tRNA1Lys to Escherichia coli ribosomes was stimulated with ApApA as well as ApApG.     

Genome-wide analysis of N1-methyl-adenosine modification in human tRNAs

The N¹-methyl-Adenosine (m¹A58) modification at the conserved nucleotide 58 in the TΨC loop is present in most eukaryotic tRNAs. In yeast, m¹A58 modification is essential for viability because it is required for the stability of the initiator-tRNA^Met. However, m¹A58 modification is not required for the stability of several other tRNAs in yeast. This differential m¹A58 response for different tRNA species raises the question of whether some tRNAs are hypomodified at A58 in normal cells, and how hypomodification at A58 may affect the stability and function of tRNA. Here, we apply a genomic approach to determine the presence of m¹A58 hypomodified tRNAs in human cell lines and show how A58 hypomodification affects stability and involvement of tRNAs in translation. Our microarray-based method detects the presence of m¹A58 hypomodified tRNA species on the basis of their permissiveness in primer extension. Among five human cell lines examined, approximately one-quarter of all tRNA species are hypomodified in varying amounts, and the pattern of the hypomodified tRNAs is quite similar. In all cases, no hypomodified initiator-tRNA^Met is detected, consistent with the requirement of this modification in stabilizing this tRNA in human cells. siRNA knockdown of either subunit of the m¹A58-methyltransferase results in a slow-growth phenotype, and a marked increase in the amount of m¹A58 hypomodified tRNAs. Most m¹A58 hypomodified tRNAs can associate with polysomes in varying extents. Our results show a distinct pattern for m¹A58 hypomodification in human tRNAs, and are consistent with the notion that this modification fine tunes tRNA functions in different contexts.     

Specific interaction between anticodon nuclease and the tRNA(Lys) wobble base

The bacterial tRNA(Lys)-specific PrrC-anticodon nuclease cleaves its natural substrate 5' to the wobble base, yielding 2',3'-cyclic phosphate termini. Previous work has implicated the anticodon of tRNA(Lys) as a specificity element and a cluster of amino acid residues at the carboxy-proximal half of PrrC in its recognition. We further examined these assumptions by assaying unmodified and hypomodified derivatives of tRNA(Lys) as substrates of wild-type and mutant alleles of PrrC. The data show, first, that the anticodon sequence and wobble base modifications of tRNA(Lys) play major roles in the interaction with anticodon nuclease. Secondly, a specific contact between the substrate recognition site of PrrC and the tRNA(Lys) wobble base is revealed by PrrC missense mutations that suppress the inhibitory effects of wobble base modification mutations. Thirdly, the data distinguish between the anticodon recognition mechanisms of PrrC and lysyl-tRNA synthetase.