FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2025121505> ?p ?o ?g. }

• W2025121505 endingPage "1134" @default.
• W2025121505 startingPage "1122" @default.
• W2025121505 abstract "Article1 March 1997free access The ‘polysemous’ codon—a codon with multiple amino acid assignment caused by dual specificity of tRNA identity Tsutomu Suzuki Tsutomu Suzuki Department of Biological Sciences, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama, 227 Japan Search for more papers by this author Takuya Ueda Corresponding Author Takuya Ueda Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113 Japan Search for more papers by this author Kimitsuna Watanabe Corresponding Author Kimitsuna Watanabe Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113 Japan Search for more papers by this author Tsutomu Suzuki Tsutomu Suzuki Department of Biological Sciences, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama, 227 Japan Search for more papers by this author Takuya Ueda Corresponding Author Takuya Ueda Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113 Japan Search for more papers by this author Kimitsuna Watanabe Corresponding Author Kimitsuna Watanabe Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113 Japan Search for more papers by this author Author Information Tsutomu Suzuki2, Takuya Ueda 1 and Kimitsuna Watanabe 1 1Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113 Japan 2Department of Biological Sciences, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama, 227 Japan The EMBO Journal (1997)16:1122-1134https://doi.org/10.1093/emboj/16.5.1122 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In some Candida species, the universal CUG leucine codon is translated as serine. However, in most cases, the serine tRNAs responsible for this non-universal decoding (tRNASerCAG) accept in vitro not only serine, but also, to some extent, leucine. Nucleotide replacement experiments indicated that m1G37 is critical for leucylation activity. This finding was supported by the fact that the tRNASerCAGs possessing the leucylation activity always have m1G37, whereas that of Candida cylindracea, which possesses no leucylation activity, has A37. Quantification of defined aminoacetylated tRNAs in cells demonstrated that 3% of the tRNASerCAGs possessing m1G37 were, in fact, charged with leucine in vivo. A genetic approach using an auxotroph mutant of C.maltosa possessing this type of tRNASerCAG also suggested that the URA3 gene inactivated due to the translation of CUG as serine was rescued by a slight incorporation of leucine into the polypeptide, which demonstrated that the tRNA charged with multiple amino acids could participate in the translation. These findings provide the first evidence that two distinct amino acids are assigned by a single codon, which occurs naturally in the translation process of certain Candida species. We term this novel type of codon a ‘polysemous codon’. Introduction The universality of the genetic code was once considered to be one of the essential characteristics of life, which led to the conception of the ‘frozen accident theory’. This theory proposes that all extant living organisms use the universal genetic code, which was born by accident and ‘frozen’, and that they originate from a single, closely interbreeding population (Crick, 1968). However, in recent years a number of non-universal genetic codes have been reported in various non-plant mitochondrial systems, as well as in several nuclear systems (reviewed in Osawa et al., 1992; Osawa, 1995), which contradict the frozen accident theory. Among these deviations from the universal codes, Kawaguchi et al. (1989) demonstrated that CUG, a universal leucine codon, is translated as serine in an asporogenic yeast, Candida cylindracea. We identified the serine tRNA having the anticodon CAG, which is responsible for the assignment of codon CUG as serine (termed tRNASerCAG), and revealed its decoding mechanism by means of an in vitro translational assay system (Yokogawa et al., 1992; Suzuki et al., 1994). Furthermore, when we investigated the distribution of this non-universal genetic code in fungi, as well as C.cylindracea, eight other Candida species—C.albicans, C.zeylanoides, C.lusitaniae, C.tropicalis, C.melbiosica, C.parapsilosis, C.guilliermondii and C.rugosa—were found to utilize the codon CUG for serine instead of leucine, all having tRNASerCAG as the mediator in the unusual decoding (Ohama et al., 1993; Ueda et al., 1994). Several other investigators have also shown that the codon CUG is actually translated as serine in vivo in C.albicans and C.maltosa (Santos and Tuite, 1995a; Sugiyama et al., 1995; Zimmer and Schunck, 1995). One of the most remarkable structural features observed in most of these tRNASerCAGs is that the nucleotide 5′-adjacent to the anticodon (position 33) is occupied not by the conserved U residue (U33) but by a G residue (G33). It has been speculated that U33 is necessary for forming the U-turn structure of the anticodon loop in all tRNAs reported so far (Quigley and Rich, 1976; Sprinzl et al., 1996). Moreover, the nucleotide at position 37, 3′-adjacent to the anticodon CAG, is 1-methyl guanosine (m1G) in almost all tRNASerCAGs except for that of C.cylindracea (A37), while all the serine tRNAs in fungi corresponding to the universal serine codons UCN (N: A, C, U or G) and AGY (Y: U or C) have modified adenosine at this position without exception (Sprinzl et al., 1996). The question then arises as to why these tRNASerCAGs specific for this non-universal codon possess such unique features in the sequence surrounding the anticodon. In the past 10 years, the mechanism by which aminoacyl-tRNA synthetases recognize their cognate tRNAs has been extensively investigated both in vitro and in vivo (Schimmel, 1987; Schulman and Abelson, 1988; Yarus, 1988; Normanly and Abelson, 1989; Shimizu et al., 1992; McClain, 1993; Schimmel et al., 1993). This line of study began with the artificial conversion of leucine tRNA of Escherichia coli to serine tRNA by Abelson's group 10 years ago (Normanly et al., 1986). Recently, tRNA identity elements of Saccharomyces cerevisiae leucine tRNA were elucidated using unmodified variants synthesized by T7 RNA polymerase (Soma et al., 1996), indicating that in addition to the discriminator base, A73, the second letter of the anticodon, A35, and the nucleotide 3′-adjacent to the anticodon, m1G37, are important for recognition by leucyl-tRNA synthetase (LeuRS). The majority of Candida tRNASerCAGs have A35 and m1G37, while the discriminator is occupied by a nucleoside other than adenosine (mostly G73). In this respect, tRNASerCAG seems to be a potentially chimeric tRNA molecule capable of being recognized not only by seryl- but also by leucyl-tRNA synthetases. Previously, we showed that these tRNASerCAGs would have originated from the serine tRNA corresponding to codon UCG (Ueda et al., 1994). This suggests an evolutionary pathway in which conversion from A to m1G would have taken place at position 37 just after the emergence of tRNASerCAG had brought about a change in the universal code. Since such a mutation at position 37 might potentially result in the leucylation of tRNASerCAG, we attempted to elucidate the charging properties of these tRNASerCAGs both in vitro and in vivo. Based on the results of in vitro aminoacylation reactions using tRNA variants constructed by the microsurgery method, the direct analysis of aminoacylated tRNAs in cells and a genetic approach, we demonstrate here that these serine tRNAs are actually leucylated both in vitro and in vivo. Furthermore, m1G at position 37 was found to be indispensable for the leucylation of tRNASerCAGs. In fact, the tRNASerCAG of C.cylindracea, which has A at position 37, exhibits no leucylation activity. C.cylindracea has a high G+C content (63%) and utilizes CUG as a major serine codon. However, the other Candida species have no such high G+C content and utilize the CUG as a minor serine codon (Kawaguchi et al., 1989; Lloyd and Sharp, 1992; our unpublished observation). Considering the relationship between the usage of the codon CUG as serine and the leucylation properties of tRNASerCAG, it seems that only Candida species with a genome in which the incidence of the CUG serine codon is very low possess serine tRNASerCAG that can be leucylated. Furthermore, such tRNASerCAGs charged with heterogeneous amino acids should be utilized equally in the translation process. This is the first demonstration that a single tRNA species is assigned to two different amino acids in the cell. We propose designating this type of codon having multiple amino acid assignment as a ‘polysemous codon’. The correlation between the dual-assignment state and the pathway of genetic code diversification is also discussed. Results Candida zeylanoides tRNASerCAG is leucylated in vitro First the leucylation of tRNASerCAGs from C.zeylanoides and C.cylindracea was examined using LeuRS partially purified from C.zeylanoides, since it is known that leucine tRNAs of yeast have one of their identity determinants at position 37 (Soma et al., 1996) and tRNASerCAGs of C.zeylanoides and C.cylindracea have different nucleotides at this position (m1G and A, respectively) (Figure 1A). Both tRNAs showed almost full serylation activity (∼1200–1500 pmol/A260 unit), as shown in Figure 1B. The tRNASerCAG of C.zeylanoides was evidently leucylated (the kinetic parameters are given in the uppermost row of Table I), though the charging activity was lower than that for serylation. This low acceptance of leucine of tRNASerCAG may be due to the partial purification of LeuRS and high Km value of LeuRS towards tRNASerCAG. On the contrary, tRNASerCAG of C.cylindracea was not leucylated at all, as was the case when another species of serine tRNA specific for codon AGY (Y: U or C) (tRNASerGCU) was employed as a control substrate (Figure 1B, right-hand graph). The Km value of C.zeylanoides LeuRS towards tRNASerCAG (5.0 μM) is only one order of magnitude larger than that of the serylation of this tRNA (0.22 μM) as well as that of leucylation toward the cognate leucine tRNAs of S.cerevisae (0.34 μM; Soma et al., 1996). Figure 1.Aminoacylation of Candida tRNASerCAGs with serine and leucine. (A) Cloverleaf structures of tRNASerCAGs from C.zeylanoides and C.cylindracea (Yokogawa et al., 1992; Ohama et al., 1993). The numbering system and abbreviations for modified nucleotides conform to Sprinzl et al. (1996) and Crain and McCloskey (1996), respectively. (B) Time-dependent aminoacylation with SerRS or LeuRS from C.zeylanoides cells. Aminoacylation reactions were carried out with 0.7 μM tRNAs and with same amounts of enzyme activities calculated using cognate tRNAs. Serylation and leucylation are shown by dotted and solid lines, respectively. The right-hand frame shows the solid curves from left-hand frame plotted with an enlarged ordinate. The aminoacylation of C.zeylanoides tRNASerCAG (○) and of C.cylindracea tRNASerCAG (□) are compared; C.cylindracea tRNASerGCU (▪), having no leucylation activity, is shown as a control. (C) TLC analysis of acetylleucyl-tRNA fragments derived from leucylated tRNASerCAGs. After leucylation with [14C]leucine, leucyl-tRNAs were acetylated with acetic anhydride. Acetyl-[14C]leucyl-tRNASerCAG of C.zeylanoides digested with RNase T1 (lane 1), and acetyl-[14C]leucyl-tRNALeus digested with RNase U2 (lane 3) or RNase T1 (lane 4) were developed on a TLC plate. Lane 2 is the pattern developed using a mixed sample from lanes 1 and 3. Samples containing radioactivity of 200 c.p.m. were spotted onto a cellulose TLC plate and developed by saturated ammonium sulfate/1 M NaOAc (pH 5.5)/isopropanol/dH2O(20/9/1/20). The radioactivities were visualized by an imaging analyzer (BAS-1000, Fuji Photo Systems). Download figure Download PowerPoint Figure 2.Construction of tRNA variants with mutation at position 33 or 37 by the microsurgery method. (A) Sequences of the anticodon region of variants of tRNASerCAGs from C.zeylanoides and C.cylindracea. The mutated nucelotides are shown by white letters on a black background. (B) Gel electrophoretic patterns showing RNA sequences around the anticodon for two variants mutated at position 37 (m1G37A and m1G37G) by Donis-Keller's method (Donis-Keller, 1980). −E, Al, T1, U2, PhyM and CL3 indicate no treatment and alkaline digestion, RNase T1 (specific for G), RNase U2 (for A > G), RNase PhyM (for A and U) and RNase CL3 (for C) treatments, respectively. Bands corresponding to A or G appeared at position 37 in m1G37A or m1G37G, respectively, while none was observed in the native tRNA (shown by arrows). (C) Gel electrophoretic patterns showing RNA sequences around the anticodon for variants mutated at position 33 by Donis-Keller's method. The nucleotide at position 33 in each variant was confirmed to have been replaced as expected (shown by arrows). Download figure Download PowerPoint Table 1. Kinetic parameters for mutants of tRNASerCAG from C.zeylanoides with leucyl-tRNA synthetase of C.zeylanoides Strain Km (μM)a Vmax (pmol/min) Vmax/Km (relative) Native 5.0 3.3 1.0 z-G33C 1.3 3.1 3.8 z-G33U 1.4 1.2 1.3 z-G33G 5.6 2.2 0.59 z-G33A 6.7 2.5 0.56 a The apparent Km values are given since the LeuRS used was a partially purified fraction. In order to verify that the leucylation activity observed for the tRNASerCAG of C.zeylanoides actually came from the tRNASerCAG itself, and not from a trace amount of leucine tRNA contaminating the tRNA sample, the leucylated 3′-terminal RNA fragment derived from leucyl-tRNASerCAG was analyzed in the following manner. 14C-leucylated tRNASerCAG from C.zeylanoides was first acetylated with acetic anhydride to prevent deacylation, and then digested with RNase T1. The resulting 3′-terminal fragment with 14C-labeled acetylleucine was analyzed by cellulose TLC. The results are shown in Figure 1C. If leucylated tRNASerCAG were digested with RNase T1, 14C-labeled acetylleucyl-CCA should be released as a labeled fragment (Figure 1C, lane 3), because G is located at position 73 of the tRNASerCAG (Figure 1A, left-hand structure). Any contaminated leucine tRNAs, if they exist, will give some 14C-labeled fragments larger than the tetramer (Figure 1C, lane 4), because all the leucine tRNAs of yeasts so far analyzed (Sprinzl et al., 1996) including those of C.zeylanoides (T.Suzuki, unpublished result) are known to have A73 at their 3′-ends, which are resistant to RNase T1. The mobility of the acetylleucyl-oligonucleotide derived from tRNASerCAG from C.zeylanoides (Figure 1C, lane 1) was identical to that of acelylleucyl-CCA prepared from the RNase U2 digests of leucyl-tRNALeus from C.zeylanoides (lane 3). This observation clearly demonstrates that leucine is definitely attached to the tRNA possessing G73; the tRNA therefore must be tRNASerCAG and not tRNALeu. Thus, it is concluded that the tRNA which incorporated leucine in vitro is in fact tRNASerCAG. This deduction is supported by the results of an additional experiment: incorporation of [14C]leucine into the tRNASerCAG sample with LeuRS was reduced by the addition of SerRS and non-labeled serine to the reaction mixture (data not shown), which clearly indicates that the same tRNA molecule is competitively aminoacylated by these two enzymes. To conclude that tRNASerCAG is aminoacylated with leucine, we carried out a further experiment. The tRNASerCAG was charged with serine and serylated tRNASerCAG was separated from non-aminoacylated tRNASerCAG by gel-electrophoresis under acidic conditions. After deacylation, the leucylation activity of the tRNASerCAG was unequivocally detected. This experiment clearly indicates that a tRNASerCAG molecule with serylation activity simultaneously possesses leucine-accepting activity. Leucyl-tRNA synthetase from C.zeylanoides also leucylated tRNASerCAGs from C.albicans, C.lusitaniae and C.tropicalis (data not shown), but the tRNASerCAG of C.cylindracea was not leucylated at all. This charging property was not due to the heterologous combination of the synthetase and tRNA, since similar results were observed with LeuRSs from both C.cylindracea and S.cerevisiae (data not shown). m1G37 is responsible for recognition by leucyl-tRNA synthetase Among the tRNASerCAGs of several Candida species, that of C.cylindracea is unique because it alone possesses no leucylation capacity. A sequence comparison of these tRNAs (Figure 1A) prompts us to speculate that the nucleotide at position 37 is strongly associated with leucylation, because all tRNASerCAGs possessing leucylation activity have m1G in common, while only the tRNASerCAG of C.cylindracea, which possesses no leucylation activity, has A at this position. To examine the validity of this speculation, a series of tRNASerCAG variants was constructed by the in vitro transcription method using T7 RNA polymerase, as well as by the microsurgery method, and the leucylation activity of each variant was measured. When the tRNASerCAG of C.zeylanoides synthesized by in vitro transcription was employed as a substrate, no leucylation activity was detected, not even for the tRNA transcript having G37 (Figure 3A). On the other hand, as shown in Figure 3A, serylation activity exceeded 1000 pmol/A260unit. These results strongly suggested that some nucleoside modification is necessary in tRNASerCAG for recognition by LeuRS. We thus attempted to replace the m1G37 of C.zeylanoides tRNASerCAG with G (the variant is symbolized as m1G37G) or A (m1G37A), by the microsurgery method (Figure 2A and B; for details, see Materials and methods) to examine the contribution of m1G37 to leucylation and the contribution of A37 of C.cylindracea tRNASerCAG to the prevention of leucylation. Figure 3.Aminoacylation of tRNA variants with serine and leucine. (A) Effect of replacement of m1G37 on leucylation. Variants: C.zeylanoides tRNASerCAG (●), C.cylindracea tRNASerCAG (▴), m1G37A (□), m1G37G (▪), z-G33G (○) and the transcript of C.zeylanoides tRNASerCAG (▵). Solid and dotted lines indicate leucylation and serylation, respectively [and also in (B) and (C)]. The right-hand frame shows the solid curves from the left-hand frame plotted with an enlarged ordinate [and also in (C)]. (B) Effect of the replacement of G33 of C.zeylanoides tRNASerCAG on leucylation. Variants: z-G33C (□), z-G33U (○), z-G33G (▪) and z-G33A (●). (C) No leucylation was observed in any variant from C.cylindracea tRNASerCAG with a mutation at position 33. Variants: c-G33C (□), c–G33U (○), c-G33G (▪) and c-G33A (●). Download figure Download PowerPoint When aminoacylation of m1G37A and m1G37G was examined (Figure 3A), the results indicated that both substitutions lead to complete loss of leucylation (Figure 3A, right-hand graph), although no apparent influence was observed on serylation (Figure 3A, left-hand graph). These findings strongly indicate that the methyl group of m1G37 plays a crucial role in enhancing the leucylation activity of tRNASerCAG. The slight reduction in leucylation activity observed in the control variant z-G33G (Figure 2A) compared with native tRNA (Figure 3A, right-hand graph) was found to have resulted from the partial deacetylation of 4-acetyl cytidine (ac4C) due to acid treatment of the 5′-half fragment of tRNASerCAG (see Materials and methods). This is considered further in the Discussion. G33 acts as a modulator of leucylation In addition to m1G37, another unique feature of the serine tRNASerCAGs in these Candida species is the presence of G at position 33, where a pyrimidine (mostly U) is completely conserved in usual tRNAs (Sprinzl et al., 1996). Since we considered it is possible that this notable feature may be in some way related to the unusual aminoacylation characteristics described above and/or to the translation of non-universal genetic code, we examined the effect of residue 33 on the aminoacylation and translation activities of mutated tRNASerCAG by introducing a point mutation at this position in the tRNAs of C.zeylanoides and C.cylindracea using the microsurgery method (see Figures 2A and C, and 7B and C). The effect of mutation at position 33 in these two tRNAs was found to be quite different. In the case of the C.cylindracea tRNA, none of the mutations at position 33 caused leucylation of the tRNA, as was observed with the native tRNASerCAG, and there was no reduction in serylation activity (Figure 3C). In contrast, the replacement of G33 by pyrimidines in C.zeylanoides tRNASerCAG considerably enhanced the leucylation activity (Figure 3B, right-hand graph), while no significant difference was observed in the serylation activity (Figure 3B, left-hand graph). The kinetic parameters of leucylation for the variants of C.zeylanoides tRNA are shown in Table I. It is notable that the Km values of the two pyrimidine mutants, z-G33U (1.4 μM) and z-G33C (1.3 μM), are clearly lower than those of the two purine mutants, z-G33A (6.7 μM) and z-G33G (5.6 μM). The Vmax value of z-G33U (1.2 pmol/min) is 39% of that of z-G33C (3.1 pmol/min), which could explain why z-G33U shows lower leucylation activity than z-G33C despite having nearly the same Km value (Figure 3B, right-hand graph). Judging from the sequence analysis (data not shown), the slight reduction in the leucylation of z-G33G (5.6 μM) compared with that of the native tRNASerCAG (5.0 μM) is probably due to the partial deacetylation of ac4C at position 12, as mentioned above. This was confirmed by the observation of a slight reduction in leucylation activity also in acid-treated native tRNASerCAG (data not shown). It is thus concluded that replacement of a pyrimidine by a purine at position 33 has a repressive effect on leucylation of the tRNASerCAG of C.zeylanoides. The translation efficiencies of the variants with a mutation at position 33 were also examined in a cell-free translation system of C.cylindracea (Yokogawa et al., 1992; Suzuki et al., 1994), to evaluate the effect of G33. A change from G to U at position 33 apparently enhanced the translation activity 2.5-fold, although their decoding properties did not change at all (data not shown). We thus consider that G33 serves as a modulator of leucylation of tRNASerCAG, despite a slight disadvantage in translation activity. Evidence for leucylation of C.zeylanoides tRNASerCAG in vivo At this point, we had established that the tRNASerCAG of C.zeylanoides is actually able to accept leucine in vitro. However, considering the facts that SerRS and LeuRS coexist in cells and, judging from their Km values, that the affinity of tRNASerCAG toward SerRS is one order of magnitude higher than that toward LeuRS, we needed to ascertain whether the tRNASerCAG of C.zeylanoides is in fact leucylated in vivo. For this purpose, we adopted a newly developed method for quantifying an individual aminoacyl-tRNA in cells (Suzuki et al., 1996). Aminoacyl-tRNAs separately prepared from cells of C.zeylanoides and C.cylindracea were immediately subjected to acetylation using [1-14C]acetic anhydride to label the amino acids as well as to stabilize the aminoacylated tRNAs. From each of the acetylated aminoacyl-tRNA mixtures, tRNASerCAGs from C.zeylanoides and C.cylindracea were fished out by a solid-phase-attached DNA probe as described previously (Tsurui et al., 1994; Wakita et al., 1994). A single band for each of the aminoacyl-tRNAs was detected by staining (Figure 4A) with which the radioactivity coincided in each case (Figure 4B). Figure 4.Identification on TLC plates of amino acids attached to tRNASerCAGs obtained from cells of C.cylindracea and C.zeylanoides. (A) [14C]acetylaminoacyl-tRNASerCAGs purified on 10% PAGE stained by toluidine blue. Lanes: uncharged tRNASerCAG (lane 1) and [14C]acetylaminoacyl-tRNASerCAG (lane 2) from C.cylindracea, and uncharged tRNASerCAG (lane 4) and [14C]acetylaminoacyl-tRNASerCAG (lane 3) from C.zeylanoides. Unfractionated tRNA from C.zeylanoides is also shown (lane 5). (B) Autoradiograph of (A). [14C]acetylaminoacyl-tRNAs were visualized by an imaging analyzer. Lanes correspond to these in (A). (C) Identification of acetylamino acids attached to tRNASerCAGs analyzed by TLC. Lanes 2 and 3 show the spots corresponding to acetylated amino acids released from C.zeylanoides tRNASerCAG (lane 2) and C.cylindracea tRNASerCAG (lane 3) with alkaline treatment. Lanes 1 and 4 show the spots corresponding to acetylleucine and acetylserine as markers, respectively. (D) Analysis of acetylamino acids attached to tRNA fragments on a TLC plate. Lane 2 shows the spot corresponding to the acetylamino acids derived from the RNase T1 fragment of C.zeylanoides tRNASerCAG. Lanes 1 and 3 indicate the spots corresponding to acetylleucine and acetylserine, respectively. Ten micrograms of [14C]acetylaminoacyl-tRNASerCAG from C.zeylanoides was digested with RNase T1 and developed on cellulose TLC plates under the same conditions as (C). CCA fragments with [14C]acetylamino acids were scraped from the plate from which the fragments were eluted with H20 and desalted by Sep-pak C18 under the conditions described in the literature (Wang et al., 1990). [14C]acetylamino acids discharged from the fragments were developed on TLC and visualized by an imaging analyzer (BAS-1000, Fuji Photo Systems). Download figure Download PowerPoint Acetylated amino acids attached to these tRNAs were deacylated by alkaline treatment and analyzed by TLC. As shown in Figure 4C, acetylserine was observed as a major amino acid derivative in both tRNASerCAGs, but acetylleucine was detected only in the C.zeylanoides tRNASerCAG; the acetylserine and acetylleucine spots were identified as described previously (Suzuki et al., 1996). The radioactivities remaining on the origins probably came from the direct acetylation of some nucleotides in the tRNAs, as discussed previously (Suzuki et al., 1996). From comparison with the radioactivity of acetylserine, it was calculated that ∼3% of the tRNASerCAG was attached with acetylleucine. These results were reproducible. Digestion of purified acetyl-aminoacyl tRNASerCAG with RNase T1 also gave only a 14C-labeled CCA fragment, as shown in Figure 1C. When the acetylated amino acid released from the fragment purified from the corresponding spot on TLC was analyzed by TLC, the ratio of acetylleucine to acetylserine was also found to be 3% (Figure 4D), indicating that acetylleucine is covalently attached to the tRNASerCAG fragment with G73. It thus became clear that the tRNASerCAG of C.zeylanoides was in fact charged with leucine by 3% of the amount of serylation of the same tRNASerCAG in C.zeylanoides cells. Incorporation of leucine is dependent on the CUG codon in C.maltosa Aminoacylation has generally been considered to be the final stage determining translational accuracy (reviewed in Parker, 1989; Kurland, 1992; Farabaugh, 1993). However, in the case of tRNAGln charged with glutamate in the chloroplast, Glu-tRNAGln is rejected by an elongation factor so that the chloroplast translation machinery does not employ the mischarged aminoacyl-tRNA (Stanzel et al., 1994). It is likely that this is an exceptional case due to the lack of glutamyl-tRNA synthetase in the chloroplast. In order to prove that leucylated tRNASerCAGs actually participate in the translation process in Candida cells without such a rejection mechanism, we utilized a URA3 gene expression system derived from S.cerevisiae in C.maltosa, which was developed by Sugiyama et al. (1995). Candida maltosa utilizes the codon CUG as serine and possesses the relevant tRNASerCAG gene (Sugiyama et al., 1995; Zimmer and Schunck, 1995). Since the tRNASerCAG gene has G at position 37, G37 should be modified to m1G in tRNA, and tRNASerCAG may hence become chargeable with leucine in addition to serine in C.maltosa cells. In the URA3 gene of S.cerevisiae, only one CTG codon appears, at the 45th position, and this is translated as leucine according to the universal genetic code, which is essential for the activity of orotidine 5′-monophosphate decarboxylase (ODCase) (Rose et al., 1984; Sugiyama et al., 1995). In the present study, this URA3 gene, with the CTG codon replaced by various leucine or serine codons, was utilized as a marker gene (Figure 5A). First, a plasmid in which the S.cerevisiae URA3 gene was inserted downstream of a C.maltosa-specific promoter (C-p) was constructed and designated as pCSU–CTG (Sugiyama, 1995). As controls, mutant plasmids of pCSU–CTG, in which the codon CTG was replaced by either the serine codon TCT or the leucine codon CTC, were constructed and named pCSU–TCT and pCSU–CTC, respectively. In addition, a plasmid (pCCU) consisting of the URA3 gene of C.maltosa having a CTT leucine codon at the corresponding site, combined with the C.maltosa-specific promoter, was also used as a positive control. These variant plasmids were introduced into a URA3-defective C.maltosa strain CHU1 (his5, ade1, ura3::C-ADE1/ura3::C-ADE1) (Ohkuma et al., 1993), the growth of which was monitored on minimal medium SD plates in the presence and absence of uracil. Figure 5.Complementation of C.maltosa URA3 mutation by S.cerevisiae URA3 variants. (A) Construction of URA3 genes transformed into C.maltosa URA3 mutant. The" @default.
• W2025121505 created "2016-06-24" @default.
• W2025121505 creator A5001653266 @default.
• W2025121505 creator A5065130731 @default.
• W2025121505 creator A5083952135 @default.
• W2025121505 date "1997-03-01" @default.
• W2025121505 modified "2023-10-13" @default.
• W2025121505 title "The `polysemous' codon_a codon with multiple amino acid assignment caused by dual specificity of tRNA identity" @default.
• W2025121505 cites W1484204974 @default.
• W2025121505 cites W1509188194 @default.
• W2025121505 cites W1512267178 @default.
• W2025121505 cites W1550695177 @default.
• W2025121505 cites W1774953805 @default.
• W2025121505 cites W1827567297 @default.
• W2025121505 cites W1919707350 @default.
• W2025121505 cites W1925116194 @default.
• W2025121505 cites W1947722128 @default.
• W2025121505 cites W1955465764 @default.
• W2025121505 cites W1964289666 @default.
• W2025121505 cites W1964721756 @default.
• W2025121505 cites W1969002144 @default.
• W2025121505 cites W1969737313 @default.
• W2025121505 cites W1972237569 @default.
• W2025121505 cites W1973363547 @default.
• W2025121505 cites W1974515928 @default.
• W2025121505 cites W1974959097 @default.
• W2025121505 cites W1979753304 @default.
• W2025121505 cites W1980831280 @default.
• W2025121505 cites W1986028974 @default.
• W2025121505 cites W1986171929 @default.
• W2025121505 cites W1986973053 @default.
• W2025121505 cites W1989744507 @default.
• W2025121505 cites W1993419746 @default.
• W2025121505 cites W1994207191 @default.
• W2025121505 cites W1995143868 @default.
• W2025121505 cites W2000111317 @default.
• W2025121505 cites W2002036062 @default.
• W2025121505 cites W2003148658 @default.
• W2025121505 cites W2010299178 @default.
• W2025121505 cites W2018472585 @default.
• W2025121505 cites W2026350792 @default.
• W2025121505 cites W2026615833 @default.
• W2025121505 cites W2028528516 @default.
• W2025121505 cites W2030004023 @default.
• W2025121505 cites W2039159054 @default.
• W2025121505 cites W2042161296 @default.
• W2025121505 cites W2043305143 @default.
• W2025121505 cites W2043406399 @default.
• W2025121505 cites W2045251366 @default.
• W2025121505 cites W2046884078 @default.
• W2025121505 cites W2048284469 @default.
• W2025121505 cites W2051214116 @default.
• W2025121505 cites W2053845753 @default.
• W2025121505 cites W2056570096 @default.
• W2025121505 cites W2080851619 @default.
• W2025121505 cites W2081645231 @default.
• W2025121505 cites W2083003129 @default.
• W2025121505 cites W2083812890 @default.
• W2025121505 cites W2084207316 @default.
• W2025121505 cites W2085029536 @default.
• W2025121505 cites W2086008885 @default.
• W2025121505 cites W2091095027 @default.
• W2025121505 cites W2092221199 @default.
• W2025121505 cites W2111778812 @default.
• W2025121505 cites W2127543611 @default.
• W2025121505 cites W2132897827 @default.
• W2025121505 cites W2138840910 @default.
• W2025121505 cites W2157226738 @default.
• W2025121505 cites W2167673850 @default.
• W2025121505 cites W2173055149 @default.
• W2025121505 cites W2174000974 @default.
• W2025121505 cites W2178409878 @default.
• W2025121505 cites W306515700 @default.
• W2025121505 doi "https://doi.org/10.1093/emboj/16.5.1122" @default.
• W2025121505 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1169711" @default.
• W2025121505 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9118950" @default.
• W2025121505 hasPublicationYear "1997" @default.
• W2025121505 type Work @default.
• W2025121505 sameAs 2025121505 @default.
• W2025121505 citedByCount "127" @default.
• W2025121505 countsByYear W20251215052012 @default.
• W2025121505 countsByYear W20251215052013 @default.
• W2025121505 countsByYear W20251215052014 @default.
• W2025121505 countsByYear W20251215052015 @default.
• W2025121505 countsByYear W20251215052016 @default.
• W2025121505 countsByYear W20251215052017 @default.
• W2025121505 countsByYear W20251215052018 @default.
• W2025121505 countsByYear W20251215052019 @default.
• W2025121505 countsByYear W20251215052020 @default.
• W2025121505 countsByYear W20251215052021 @default.
• W2025121505 countsByYear W20251215052022 @default.
• W2025121505 countsByYear W20251215052023 @default.
• W2025121505 crossrefType "journal-article" @default.
• W2025121505 hasAuthorship W2025121505A5001653266 @default.
• W2025121505 hasAuthorship W2025121505A5065130731 @default.
• W2025121505 hasAuthorship W2025121505A5083952135 @default.
• W2025121505 hasBestOaLocation W20251215051 @default.
• W2025121505 hasConcept C104317684 @default.

• next