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W1979201731 abstract "In the starfish mitochondrial (mt) genome, codons AGA and AGG (in addition to AGU and AGC) have been considered to be translated as serine. There is, however, only a single candidate mt tRNA gene responsible for translating these codons and it has a GCT anticodon sequence, but guanosine at the first position of the anticodon should base pair only with pyrimidines according to the conventional wobble rule. To solve this enigma, the mt tRNAGCUser was purified, and sequence determination in combination with electrospray liquid chromatography/mass spectrometry revealed that 7-methylguanosine is located at the first position of the anticodon. This is the first case in which a tRNA has been found to have 7-methylguanosine at the wobble position. It is suggested that methylation at N-7 of wobbling guanosine endows the tRNA with the capability of forming base pairs with all four nucleotides, A, U, G, and C, and expands the repertoire of codon-anticodon interaction. This finding indicates that a nonuniversal genetic code in starfish has been generated by base modification in the tRNA anticodon. In the starfish mitochondrial (mt) genome, codons AGA and AGG (in addition to AGU and AGC) have been considered to be translated as serine. There is, however, only a single candidate mt tRNA gene responsible for translating these codons and it has a GCT anticodon sequence, but guanosine at the first position of the anticodon should base pair only with pyrimidines according to the conventional wobble rule. To solve this enigma, the mt tRNAGCUser was purified, and sequence determination in combination with electrospray liquid chromatography/mass spectrometry revealed that 7-methylguanosine is located at the first position of the anticodon. This is the first case in which a tRNA has been found to have 7-methylguanosine at the wobble position. It is suggested that methylation at N-7 of wobbling guanosine endows the tRNA with the capability of forming base pairs with all four nucleotides, A, U, G, and C, and expands the repertoire of codon-anticodon interaction. This finding indicates that a nonuniversal genetic code in starfish has been generated by base modification in the tRNA anticodon. An unusual mode of wobble base interaction with mRNA codons exists in mitochondria (1Osawa S. Evolution of the Genetic Code. Oxford University Press, Oxford, UK1995Google Scholar, 2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar). Uridine at the wobble position base pairs with all four nucleotides, while modification from uridine to 5-carboxymethylaminomethyluridine (cmnm5U) 1The abbreviations used are: cmnm5U, 5-carboxymethylaminomethyluridine; mt, mitochondrial; m7G, 7-methylguanosine; m5C, 5-methylcytidine; m3C, 3-methylcytidine; f5C, 5-formylcytidine; ms2t6A, 2-methylthio-N 6-threonylcarbamoyladenosine; m6t6A, N 6-methyl-N 6-threonylcarbamoyladenosine; Y, wyosine; ES, electrospray; LC, liquid chromatography; MS, mass spectrometry. 1The abbreviations used are: cmnm5U, 5-carboxymethylaminomethyluridine; mt, mitochondrial; m7G, 7-methylguanosine; m5C, 5-methylcytidine; m3C, 3-methylcytidine; f5C, 5-formylcytidine; ms2t6A, 2-methylthio-N 6-threonylcarbamoyladenosine; m6t6A, N 6-methyl-N 6-threonylcarbamoyladenosine; Y, wyosine; ES, electrospray; LC, liquid chromatography; MS, mass spectrometry. prevents pairing with pyrimidines. Codons with a pyrimidine in the third position are translated by tRNAs having unmodified guanosine at the first anticodon position. This rule was first proposed for fungal mitochondria (3Heckman J.E. Sarnoff J. Alzner-DeWeerd B. Yin S. RajBhandary U.L. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3159-3163Crossref PubMed Scopus (204) Google Scholar, 4Martin R.P. Sibler A.P. Gehrke C.W. Kuo K. Edmonds C.G. McCloskey J.A. Dirheimer G. Biochemistry. 1990; 29: 956-959Crossref PubMed Scopus (34) Google Scholar) and has been considered to hold for most codon boxes in animal mitochondria. However, a few exceptional codon-anticodon pairings have been also observed in this organelle.In most animal mitochondria, the methionine codons AUA and AUG are translated by a single methionine tRNA which has 5-formylcytidine (f5C) in the first anticodon position, indicating that f5C plays a role similar to cmnm5U (5Moriya J. Yokogawa T. Wakita K. Ueda T. Nishikawa K. Crain P.F. Hashizume T. Pomerantz S.C. McCloskey J.A. Kawai G. Hayashi N. Yokoyama S. Watanabe K. Biochemistry. 1994; 33: 2234-2239Crossref PubMed Scopus (85) Google Scholar). In starfish and sea urchin mitochondria, however, AUA is restored to the isoleucine codon as in the universal genetic code (6Himeno H. Masaki H. Kawai T. Ohta T. Kumagai I. Miura K.-I. Watanabe K. Gene (Amst.). 1987; 56: 219-230Crossref PubMed Scopus (77) Google Scholar, 7Jacobs H.T. Elliot D.J. Math V.B. Farquharson A. J. Mol. Biol. 1988; 202: 185-217Crossref PubMed Scopus (246) Google Scholar, 8Cantatore P. Roberti M. Rainaldi G. Gadaleta M.N. Saccone C. J. Biol. Chem. 1989; 264: 10965-10975Abstract Full Text PDF PubMed Google Scholar, 9Asakawa S. Himeno H. Miura K. Watanabe K. Genetics. 1995; 140: 1047-1060PubMed Google Scholar), while AAA and AGR (R = A, G) are presumably translated as lysine and serine, respectively (6Himeno H. Masaki H. Kawai T. Ohta T. Kumagai I. Miura K.-I. Watanabe K. Gene (Amst.). 1987; 56: 219-230Crossref PubMed Scopus (77) Google Scholar, 7Jacobs H.T. Elliot D.J. Math V.B. Farquharson A. J. Mol. Biol. 1988; 202: 185-217Crossref PubMed Scopus (246) Google Scholar, 8Cantatore P. Roberti M. Rainaldi G. Gadaleta M.N. Saccone C. J. Biol. Chem. 1989; 264: 10965-10975Abstract Full Text PDF PubMed Google Scholar, 9Asakawa S. Himeno H. Miura K. Watanabe K. Genetics. 1995; 140: 1047-1060PubMed Google Scholar); these codon assignments are different from those in other known animal mitochondrial systems. For example, AGR codons are utilized for the termination and glycine codon in mammalian (10Anderson S. Bankier A.T. Barrell B.G. de Bruijn M.H.L. Coulson A.R. Drouin J. Eperon I.C. Nierlich D.P. Roe B.A. Sanger F. Schreier P.H. Smith A.J.H. Staden R. Young I.G. Nature. 1981; 290: 457-465Crossref PubMed Scopus (7535) Google Scholar) and ascidian (11Yokobori S. Ueda T. Watanabe K. J. Mol. Evol. 1993; 36: 1-8Crossref PubMed Scopus (41) Google Scholar) mitochondria, respectively. In Drosophila, AGA is the serine codon but the other AGR codon, AGG, is absent from the mitochondrial genome (12de Bruijn M.H. Nature. 1983; 304: 234-241Crossref PubMed Scopus (225) Google Scholar, 13Wolstenholme D.R. Clary D.O. Genetics. 1985; 109: 725-744Crossref PubMed Google Scholar, 14Clary D.O. Wolstenholme D.R. J. Mol. Evol. 1985; 22: 252-271Crossref PubMed Scopus (1237) Google Scholar). In contrast, both AGA and AGG appear in reading frames encoded on starfish and sea urchin mt DNAs and are presumably assigned to serine judging from the sequence alignment (6Himeno H. Masaki H. Kawai T. Ohta T. Kumagai I. Miura K.-I. Watanabe K. Gene (Amst.). 1987; 56: 219-230Crossref PubMed Scopus (77) Google Scholar, 7Jacobs H.T. Elliot D.J. Math V.B. Farquharson A. J. Mol. Biol. 1988; 202: 185-217Crossref PubMed Scopus (246) Google Scholar, 8Cantatore P. Roberti M. Rainaldi G. Gadaleta M.N. Saccone C. J. Biol. Chem. 1989; 264: 10965-10975Abstract Full Text PDF PubMed Google Scholar, 9Asakawa S. Himeno H. Miura K. Watanabe K. Genetics. 1995; 140: 1047-1060PubMed Google Scholar). In nematodes, AGN codons are translated with a serine tRNA whose gene possesses a TCT anticodon sequence (15Okimoto R. Macfarlane J.L. Clary D.O. Wolstenholme D.R. Genetics. 1992; 130: 471-498Crossref PubMed Google Scholar). We have shown that unmodified uridine is located at the wobble position of the tRNA (16Watanabe Y. Tsurui H. Ueda T. Furushima R. Takamiya S. Kita K. Nishikawa K. Watanabe K. J. Biol. Chem. 1994; 269: 22902-22906Abstract Full Text PDF PubMed Google Scholar), indicating that the codon-anticodon interaction definitely follows the mitochondrial wobble rule. In contrast, the mitochondrial gene encoding the tRNA that translates all AGN codons in starfish and sea urchin has the anticodon GCT (6Himeno H. Masaki H. Kawai T. Ohta T. Kumagai I. Miura K.-I. Watanabe K. Gene (Amst.). 1987; 56: 219-230Crossref PubMed Scopus (77) Google Scholar, 7Jacobs H.T. Elliot D.J. Math V.B. Farquharson A. J. Mol. Biol. 1988; 202: 185-217Crossref PubMed Scopus (246) Google Scholar, 8Cantatore P. Roberti M. Rainaldi G. Gadaleta M.N. Saccone C. J. Biol. Chem. 1989; 264: 10965-10975Abstract Full Text PDF PubMed Google Scholar, 9Asakawa S. Himeno H. Miura K. Watanabe K. Genetics. 1995; 140: 1047-1060PubMed Google Scholar). The question that arises from this is how the GCT anticodon can recognize the AGA and AGG codons, in addition to AGY (Y = U, C), in echinoderms.We have previously speculated on possible answers to this question (1Osawa S. Evolution of the Genetic Code. Oxford University Press, Oxford, UK1995Google Scholar,2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar, 17Watanabe K. Osawa S. Soll D. RajBhandary U.L. tRNA Structure, Biosynthesis and Function. ASM Press, Washington, D. C.1995: 225-250Google Scholar). One possibility is that G at the first anticodon position of the starfish mt tRNAGCUSer might be modified so as to allow it to pair with all four AGN codons. Alternatively, it might be that a region(s) in the tRNA other than the anticodon influences the decoding ability of AGR codons, because most metazoan mt tRNAGCUSer have unusual secondary structures in which the D arm is lacking or incomplete (2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar,17Watanabe K. Osawa S. Soll D. RajBhandary U.L. tRNA Structure, Biosynthesis and Function. ASM Press, Washington, D. C.1995: 225-250Google Scholar). This truncated D arm could be responsible for G·R pairing, in addition to G·Y pairing, between the anticodon and codons. Another feature is the G·C pair at the bottom of the anticodon stem, which is present in the tRNAGCUSer of mitochondria of invertebrates, but not in those of most vertebrates, in which its place is taken by an A·U pair (2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar, 17Watanabe K. Osawa S. Soll D. RajBhandary U.L. tRNA Structure, Biosynthesis and Function. ASM Press, Washington, D. C.1995: 225-250Google Scholar, 18Sprinzl M. Steegborn C. Hübel F. Steinberg S. Nucleic Acids Res. 1996; 24: 68-72Crossref PubMed Scopus (160) Google Scholar). Since tRNAGCUSer of all metazoan mitochondria except for that of nematode have identical anticodon loops (15Okimoto R. Macfarlane J.L. Clary D.O. Wolstenholme D.R. Genetics. 1992; 130: 471-498Crossref PubMed Google Scholar), there could be some structural differences in regions other than the anticodon loop, depending on which codons, AGY (vertebrates) or AGN (N = A, G, C, or U) (invertebrates), are translated as serine.With the aim of determining which of these speculations is actually the case, the starfish mt tRNAGCUSerresponsible for the noncanonical decoding in the AGN codon box was purified and sequenced, and the modified nucleoside content was determined by electrospray liquid chromatography-mass spectrometry (ES-LC/MS). The results showed the first of the speculations posited above to be correct; guanosine at the wobble position of starfish mt tRNAGCUSer has been completely converted to 7-methylguanosine (m7G). This strongly suggests that m7G is capable of base pairing with all four nucleotides, thus resulting in a genetic code change in echinoderm mitochondria. Based on the primary structure of the mt tRNAGCUSer, we also present a scenario that could explain evolutionary changes in the genetic code that have occurred in animal mitochondria.DISCUSSIONWe report here the first known occurrence of m7G at the wobble position of a tRNA. Even since we first discovered that in starfish mitochondria AGN codons seem to be translated as serine by the tRNAGCUSer from the sole corresponding gene located on the mt genome (6Himeno H. Masaki H. Kawai T. Ohta T. Kumagai I. Miura K.-I. Watanabe K. Gene (Amst.). 1987; 56: 219-230Crossref PubMed Scopus (77) Google Scholar), the question as to how tRNAGCUSer possessing a GCU anticodon decodes all four AGN codons has attracted our interest, especially with regard to the decoding mechanism of mt tRNAs toward nonuniversal codons. This long-pending puzzle has now been solved by the finding that a modified residue (m7G) exists at the wobble position of tRNAGCUSer, which is probably responsible for decoding AGN codons. This solution seems to be the simplest one among the speculations so far postulated (see Introduction) (1Osawa S. Evolution of the Genetic Code. Oxford University Press, Oxford, UK1995Google Scholar, 2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar,17Watanabe K. Osawa S. Soll D. RajBhandary U.L. tRNA Structure, Biosynthesis and Function. ASM Press, Washington, D. C.1995: 225-250Google Scholar).We thus consider that the occurrence of m7G at the wobble position, which has emerged uniquely in echinoderm mitochondria, leads to non-canonical base pairing. This, together with the results concerning codon-anticodon relationships obtained so far for various animal mitochondria (3Heckman J.E. Sarnoff J. Alzner-DeWeerd B. Yin S. RajBhandary U.L. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3159-3163Crossref PubMed Scopus (204) Google Scholar, 4Martin R.P. Sibler A.P. Gehrke C.W. Kuo K. Edmonds C.G. McCloskey J.A. Dirheimer G. Biochemistry. 1990; 29: 956-959Crossref PubMed Scopus (34) Google Scholar, 29Barrell B.G. Anderson S. Bankier A.T. de Bruijn M.H. Chen E. Coulson A. Drouin J. Eperon I.C. Nierlich D.P. Roe B.A. Sanger F. Schreier P.H. Smith A.J. Staden R. Young I.G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3164-3166Crossref PubMed Scopus (229) Google Scholar), enables us to draw up a set of general rules for codon-anticodon interaction in mitochondria. These are summarized in Fig. 6.In conventional mitochondrial wobble rules so far elucidated (3Heckman J.E. Sarnoff J. Alzner-DeWeerd B. Yin S. RajBhandary U.L. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3159-3163Crossref PubMed Scopus (204) Google Scholar, 4Martin R.P. Sibler A.P. Gehrke C.W. Kuo K. Edmonds C.G. McCloskey J.A. Dirheimer G. Biochemistry. 1990; 29: 956-959Crossref PubMed Scopus (34) Google Scholar,29Barrell B.G. Anderson S. Bankier A.T. de Bruijn M.H. Chen E. Coulson A. Drouin J. Eperon I.C. Nierlich D.P. Roe B.A. Sanger F. Schreier P.H. Smith A.J. Staden R. Young I.G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3164-3166Crossref PubMed Scopus (229) Google Scholar), tRNAs with unmodified guanosine in the first anticodon position are considered to translate only codons terminating in a pyrimidine at the third position, while anticodons with modified uridine at the same position translate codons having a purine at the third position (Fig. 6 a).In Drosophila mitochondria, alignment of mt genes suggests that AGA is a serine codon, whereas AGG is an unassigned codon (2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar), because AGG has never appeared in Drosophila mt genomes. We have isolated Drosophila melanogastertRNAGCUSer, which presumably recognize codons AGC, AGU, and AGA (15Okimoto R. Macfarlane J.L. Clary D.O. Wolstenholme D.R. Genetics. 1992; 130: 471-498Crossref PubMed Google Scholar), and found that unmodified guanosine is the wobble nucleotide (unpublished observation). Based on the mt genomic sequence, it is unlikely that any tRNA competing with tRNAGCUSer with regard to the translation of codon AGA exists. Thus, unmodified guanosine is probably able to base pair with adenosine at the codon third position, unless a tRNA that efficiently decodes this codon (a competitor tRNA) does exist simultaneously in the translation system (Fig. 6 b).This hypothesis is supported by the following fact. In starfish mitochondria, AUA is an isoleucine codon, which differs from other known animal mitochondria in which AUA is used as a methionine codon (2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar). We found that isoleucine and methionine tRNAs of starfish have unmodified GAU and CAU anticodons, respectively (data not shown). Our observation that the wobble nucleoside of the sole mammalian mt methionine tRNA is 5-formylcytidine led us to propose the possibility that this modification allows decoding of AUA in addition to AUG (5Moriya J. Yokogawa T. Wakita K. Ueda T. Nishikawa K. Crain P.F. Hashizume T. Pomerantz S.C. McCloskey J.A. Kawai G. Hayashi N. Yokoyama S. Watanabe K. Biochemistry. 1994; 33: 2234-2239Crossref PubMed Scopus (85) Google Scholar). These codon-anticodon relationships for methionine and isoleucine in mammalian and starfish mitochondria support the above interpretation on the decoding properties of unmodified guanosine at the wobble position. Since the unmodified CAU anticodon of methionine tRNA has no capability of decoding AUA (1Osawa S. Evolution of the Genetic Code. Oxford University Press, Oxford, UK1995Google Scholar, 2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar) due to the lack of modification at the wobble position, the tRNAMet does not compete with tRNAIle in translating the AUA codon, so that tRNAIle possessing the GAU anticodon can translate not only AUC and AUU, but also AUA. Thus it can be concluded that guanosine at the wobble position decodes C, U, and A.In ascidian mitochondria, AGR codons are specific for glycine, while AGY codons are translated as serine (11Yokobori S. Ueda T. Watanabe K. J. Mol. Evol. 1993; 36: 1-8Crossref PubMed Scopus (41) Google Scholar). The corresponding mt tRNAs were isolated and sequenced; tRNAGCUSer was found to have unmodified G at the wobble position, whereas tRNAGly specific for AGR has cmnm5U at the same position (30Kondo A. Yokobori S. Ueda T. Watanabe K. Nucleic Acids Symp. Ser. 1996; 35: 279-280Google Scholar). In this case, tRNAGly may compete with tRNAGCUSer, preventing AGR codons from being mistranslated as Ser. In mammalian mitochondria, only AGU and AGC codons are translated by tRNAGCUSerpossessing the anticodon GCU, and there are no tRNAs that translate the AGA and AGG codons (1Osawa S. Evolution of the Genetic Code. Oxford University Press, Oxford, UK1995Google Scholar, 2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar). Although it is apparently in contradiction to the above-mentioned codon-anticodon rule, AGG and AGA are utilized as termination codons in mammalian mitochondria (10Anderson S. Bankier A.T. Barrell B.G. de Bruijn M.H.L. Coulson A.R. Drouin J. Eperon I.C. Nierlich D.P. Roe B.A. Sanger F. Schreier P.H. Smith A.J.H. Staden R. Young I.G. Nature. 1981; 290: 457-465Crossref PubMed Scopus (7535) Google Scholar, 31Osawa S. Ohama T. Jukes T.H. Watanabe K. J. Mol. Evol. 1989; 29: 202-207Crossref PubMed Scopus (35) Google Scholar). We propose here that a release factor (1Osawa S. Evolution of the Genetic Code. Oxford University Press, Oxford, UK1995Google Scholar, 2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar) recognizing these codons plays a role similar to that of the competitor tRNA (Fig. 6 c). Thus, only AGY codons remain for assignment to serine.The present finding that m7G is the wobble nucleotide in starfish mt tRNAGCUSer suggests that methylation of G at N7 expands codon-anticodon interaction to permit base pairing with G, in addition to A, C, and U, at the third position of the codon (Fig. 6 d). Although the influence of the altered charge distribution (resulting form N7 methylation) on the base pairing of m7G with G is unknown, we are now carrying out experiments using an in vitro translation system to confirm the decoding properties of tRNA possessing m7G at the wobble position, and also to verify the ability of m7G to base pair with all four nucleotides with respect to the structural factors. An unusual mode of wobble base interaction with mRNA codons exists in mitochondria (1Osawa S. Evolution of the Genetic Code. Oxford University Press, Oxford, UK1995Google Scholar, 2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar). Uridine at the wobble position base pairs with all four nucleotides, while modification from uridine to 5-carboxymethylaminomethyluridine (cmnm5U) 1The abbreviations used are: cmnm5U, 5-carboxymethylaminomethyluridine; mt, mitochondrial; m7G, 7-methylguanosine; m5C, 5-methylcytidine; m3C, 3-methylcytidine; f5C, 5-formylcytidine; ms2t6A, 2-methylthio-N 6-threonylcarbamoyladenosine; m6t6A, N 6-methyl-N 6-threonylcarbamoyladenosine; Y, wyosine; ES, electrospray; LC, liquid chromatography; MS, mass spectrometry. 1The abbreviations used are: cmnm5U, 5-carboxymethylaminomethyluridine; mt, mitochondrial; m7G, 7-methylguanosine; m5C, 5-methylcytidine; m3C, 3-methylcytidine; f5C, 5-formylcytidine; ms2t6A, 2-methylthio-N 6-threonylcarbamoyladenosine; m6t6A, N 6-methyl-N 6-threonylcarbamoyladenosine; Y, wyosine; ES, electrospray; LC, liquid chromatography; MS, mass spectrometry. prevents pairing with pyrimidines. Codons with a pyrimidine in the third position are translated by tRNAs having unmodified guanosine at the first anticodon position. This rule was first proposed for fungal mitochondria (3Heckman J.E. Sarnoff J. Alzner-DeWeerd B. Yin S. RajBhandary U.L. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3159-3163Crossref PubMed Scopus (204) Google Scholar, 4Martin R.P. Sibler A.P. Gehrke C.W. Kuo K. Edmonds C.G. McCloskey J.A. Dirheimer G. Biochemistry. 1990; 29: 956-959Crossref PubMed Scopus (34) Google Scholar) and has been considered to hold for most codon boxes in animal mitochondria. However, a few exceptional codon-anticodon pairings have been also observed in this organelle. In most animal mitochondria, the methionine codons AUA and AUG are translated by a single methionine tRNA which has 5-formylcytidine (f5C) in the first anticodon position, indicating that f5C plays a role similar to cmnm5U (5Moriya J. Yokogawa T. Wakita K. Ueda T. Nishikawa K. Crain P.F. Hashizume T. Pomerantz S.C. McCloskey J.A. Kawai G. Hayashi N. Yokoyama S. Watanabe K. Biochemistry. 1994; 33: 2234-2239Crossref PubMed Scopus (85) Google Scholar). In starfish and sea urchin mitochondria, however, AUA is restored to the isoleucine codon as in the universal genetic code (6Himeno H. Masaki H. Kawai T. Ohta T. Kumagai I. Miura K.-I. Watanabe K. Gene (Amst.). 1987; 56: 219-230Crossref PubMed Scopus (77) Google Scholar, 7Jacobs H.T. Elliot D.J. Math V.B. Farquharson A. J. Mol. Biol. 1988; 202: 185-217Crossref PubMed Scopus (246) Google Scholar, 8Cantatore P. Roberti M. Rainaldi G. Gadaleta M.N. Saccone C. J. Biol. Chem. 1989; 264: 10965-10975Abstract Full Text PDF PubMed Google Scholar, 9Asakawa S. Himeno H. Miura K. Watanabe K. Genetics. 1995; 140: 1047-1060PubMed Google Scholar), while AAA and AGR (R = A, G) are presumably translated as lysine and serine, respectively (6Himeno H. Masaki H. Kawai T. Ohta T. Kumagai I. Miura K.-I. Watanabe K. Gene (Amst.). 1987; 56: 219-230Crossref PubMed Scopus (77) Google Scholar, 7Jacobs H.T. Elliot D.J. Math V.B. Farquharson A. J. Mol. Biol. 1988; 202: 185-217Crossref PubMed Scopus (246) Google Scholar, 8Cantatore P. Roberti M. Rainaldi G. Gadaleta M.N. Saccone C. J. Biol. Chem. 1989; 264: 10965-10975Abstract Full Text PDF PubMed Google Scholar, 9Asakawa S. Himeno H. Miura K. Watanabe K. Genetics. 1995; 140: 1047-1060PubMed Google Scholar); these codon assignments are different from those in other known animal mitochondrial systems. For example, AGR codons are utilized for the termination and glycine codon in mammalian (10Anderson S. Bankier A.T. Barrell B.G. de Bruijn M.H.L. Coulson A.R. Drouin J. Eperon I.C. Nierlich D.P. Roe B.A. Sanger F. Schreier P.H. Smith A.J.H. Staden R. Young I.G. Nature. 1981; 290: 457-465Crossref PubMed Scopus (7535) Google Scholar) and ascidian (11Yokobori S. Ueda T. Watanabe K. J. Mol. Evol. 1993; 36: 1-8Crossref PubMed Scopus (41) Google Scholar) mitochondria, respectively. In Drosophila, AGA is the serine codon but the other AGR codon, AGG, is absent from the mitochondrial genome (12de Bruijn M.H. Nature. 1983; 304: 234-241Crossref PubMed Scopus (225) Google Scholar, 13Wolstenholme D.R. Clary D.O. Genetics. 1985; 109: 725-744Crossref PubMed Google Scholar, 14Clary D.O. Wolstenholme D.R. J. Mol. Evol. 1985; 22: 252-271Crossref PubMed Scopus (1237) Google Scholar). In contrast, both AGA and AGG appear in reading frames encoded on starfish and sea urchin mt DNAs and are presumably assigned to serine judging from the sequence alignment (6Himeno H. Masaki H. Kawai T. Ohta T. Kumagai I. Miura K.-I. Watanabe K. Gene (Amst.). 1987; 56: 219-230Crossref PubMed Scopus (77) Google Scholar, 7Jacobs H.T. Elliot D.J. Math V.B. Farquharson A. J. Mol. Biol. 1988; 202: 185-217Crossref PubMed Scopus (246) Google Scholar, 8Cantatore P. Roberti M. Rainaldi G. Gadaleta M.N. Saccone C. J. Biol. Chem. 1989; 264: 10965-10975Abstract Full Text PDF PubMed Google Scholar, 9Asakawa S. Himeno H. Miura K. Watanabe K. Genetics. 1995; 140: 1047-1060PubMed Google Scholar). In nematodes, AGN codons are translated with a serine tRNA whose gene possesses a TCT anticodon sequence (15Okimoto R. Macfarlane J.L. Clary D.O. Wolstenholme D.R. Genetics. 1992; 130: 471-498Crossref PubMed Google Scholar). We have shown that unmodified uridine is located at the wobble position of the tRNA (16Watanabe Y. Tsurui H. Ueda T. Furushima R. Takamiya S. Kita K. Nishikawa K. Watanabe K. J. Biol. Chem. 1994; 269: 22902-22906Abstract Full Text PDF PubMed Google Scholar), indicating that the codon-anticodon interaction definitely follows the mitochondrial wobble rule. In contrast, the mitochondrial gene encoding the tRNA that translates all AGN codons in starfish and sea urchin has the anticodon GCT (6Himeno H. Masaki H. Kawai T. Ohta T. Kumagai I. Miura K.-I. Watanabe K. Gene (Amst.). 1987; 56: 219-230Crossref PubMed Scopus (77) Google Scholar, 7Jacobs H.T. Elliot D.J. Math V.B. Farquharson A. J. Mol. Biol. 1988; 202: 185-217Crossref PubMed Scopus (246) Google Scholar, 8Cantatore P. Roberti M. Rainaldi G. Gadaleta M.N. Saccone C. J. Biol. Chem. 1989; 264: 10965-10975Abstract Full Text PDF PubMed Google Scholar, 9Asakawa S. Himeno H. Miura K. Watanabe K. Genetics. 1995; 140: 1047-1060PubMed Google Scholar). The question that arises from this is how the GCT anticodon can recognize the AGA and AGG codons, in addition to AGY (Y = U, C), in echinoderms. We have previously speculated on possible answers to this question (1Osawa S. Evolution of the Genetic Code. Oxford University Press, Oxford, UK1995Google Scholar,2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar, 17Watanabe K. Osawa S. Soll D. RajBhandary U.L. tRNA Structure, Biosynthesis and Function. ASM Press, Washington, D. C.1995: 225-250Google Scholar). One possibility is that G at the first anticodon position of the starfish mt tRNAGCUSer might be modified so as to allow it to pair with all four AGN codons. Alternatively, it might be that a region(s) in the tRNA other than the anticodon influences the decoding ability of AGR codons, because most metazoan mt tRNAGCUSer have unusual secondary structures in which the D arm is lacking or incomplete (2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar,17Watanabe K. Osawa S. Soll D. RajBhandary U.L. tRNA Structure, Biosynthesis and Function. ASM Press, Washington, D. C.1995: 225-250Google Scholar). This truncated D arm could be responsible for G·R pairing, in addition to G·Y pairing, between the anticodon and codons. Another feature is the G·C pair at the bottom of the anticodon stem, which is present in the tRNAGCUSer of mitochondria of invertebrates, but not in those of most vertebrates, in which its place is taken by an A·U pair (2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar, 17Watanabe K. Osawa S. Soll D. RajBhandary U.L. tRNA Structure, Biosynthesis and Function. ASM Press, Washington, D. C.1995: 225-250Google Scholar, 18Sprinzl M. Steegborn C. Hübel F. Steinberg S. Nucleic Acids Res. 1996; 24: 68-72Crossref PubMed Scopus (160) Google Scholar). Since tRNAGCUSer of all metazoan mitochondria except for that of nematode have identical anticodon loops (15Okimoto R. Macfarlane J.L. Clary D.O. Wolstenholme D.R. Genetics. 1992; 130: 471-498Crossref PubMed Google Scholar), there could be some structural differences in regions other than the anticodon loop, depending on which codons, AGY (vertebrates) or AGN (N = A, G, C, or U) (invertebrates), are translated as serine. With the aim of determining which of these speculations is actually the case, the starfish mt tRNAGCUSerresponsible for the noncanonical decoding in the AGN codon box was purified and sequenced, and the modified nucleoside content was determined by electrospray liquid chromatography-mass spectrometry (ES-LC/MS). The results showed the first of the speculations posited above to be correct; guanosine at the wobble position of starfish mt tRNAGCUSer has been completely converted to 7-methylguanosine (m7G). This strongly suggests that m7G is capable of base pairing with all four nucleotides, thus resulting in a genetic code change in echinoderm mitochondria. Based on the primary structure of the mt tRNAGCUSer, we also present a scenario that could explain evolutionary changes in the genetic code that have occurred in animal mitochondria. DISCUSSIONWe report here the first known occurrence of m7G at the wobble position of a tRNA. Even since we first discovered that in starfish mitochondria AGN codons seem to be translated as serine by the tRNAGCUSer from the sole corresponding gene located on the mt genome (6Himeno H. Masaki H. Kawai T. Ohta T. Kumagai I. Miura K.-I. Watanabe K. Gene (Amst.). 1987; 56: 219-230Crossref PubMed Scopus (77) Google Scholar), the question as to how tRNAGCUSer possessing a GCU anticodon decodes all four AGN codons has attracted our interest, especially with regard to the decoding mechanism of mt tRNAs toward nonuniversal codons. This long-pending puzzle has now been solved by the finding that a modified residue (m7G) exists at the wobble position of tRNAGCUSer, which is probably responsible for decoding AGN codons. This solution seems to be the simplest one among the speculations so far postulated (see Introduction) (1Osawa S. Evolution of the Genetic Code. Oxford University Press, Oxford, UK1995Google Scholar, 2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar,17Watanabe K. Osawa S. Soll D. RajBhandary U.L. tRNA Structure, Biosynthesis and Function. ASM Press, Washington, D. C.1995: 225-250Google Scholar).We thus consider that the occurrence of m7G at the wobble position, which has emerged uniquely in echinoderm mitochondria, leads to non-canonical base pairing. This, together with the results concerning codon-anticodon relationships obtained so far for various animal mitochondria (3Heckman J.E. Sarnoff J. Alzner-DeWeerd B. Yin S. RajBhandary U.L. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3159-3163Crossref PubMed Scopus (204) Google Scholar, 4Martin R.P. Sibler A.P. Gehrke C.W. Kuo K. Edmonds C.G. McCloskey J.A. Dirheimer G. Biochemistry. 1990; 29: 956-959Crossref PubMed Scopus (34) Google Scholar, 29Barrell B.G. Anderson S. Bankier A.T. de Bruijn M.H. Chen E. Coulson A. Drouin J. Eperon I.C. Nierlich D.P. Roe B.A. Sanger F. Schreier P.H. Smith A.J. Staden R. Young I.G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3164-3166Crossref PubMed Scopus (229) Google Scholar), enables us to draw up a set of general rules for codon-anticodon interaction in mitochondria. These are summarized in Fig. 6.In conventional mitochondrial wobble rules so far elucidated (3Heckman J.E. Sarnoff J. Alzner-DeWeerd B. Yin S. RajBhandary U.L. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3159-3163Crossref PubMed Scopus (204) Google Scholar, 4Martin R.P. Sibler A.P. Gehrke C.W. Kuo K. Edmonds C.G. McCloskey J.A. Dirheimer G. Biochemistry. 1990; 29: 956-959Crossref PubMed Scopus (34) Google Scholar,29Barrell B.G. Anderson S. Bankier A.T. de Bruijn M.H. Chen E. Coulson A. Drouin J. Eperon I.C. Nierlich D.P. Roe B.A. Sanger F. Schreier P.H. Smith A.J. Staden R. Young I.G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3164-3166Crossref PubMed Scopus (229) Google Scholar), tRNAs with unmodified guanosine in the first anticodon position are considered to translate only codons terminating in a pyrimidine at the third position, while anticodons with modified uridine at the same position translate codons having a purine at the third position (Fig. 6 a).In Drosophila mitochondria, alignment of mt genes suggests that AGA is a serine codon, whereas AGG is an unassigned codon (2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar), because AGG has never appeared in Drosophila mt genomes. We have isolated Drosophila melanogastertRNAGCUSer, which presumably recognize codons AGC, AGU, and AGA (15Okimoto R. Macfarlane J.L. Clary D.O. Wolstenholme D.R. Genetics. 1992; 130: 471-498Crossref PubMed Google Scholar), and found that unmodified guanosine is the wobble nucleotide (unpublished observation). Based on the mt genomic sequence, it is unlikely that any tRNA competing with tRNAGCUSer with regard to the translation of codon AGA exists. Thus, unmodified guanosine is probably able to base pair with adenosine at the codon third position, unless a tRNA that efficiently decodes this codon (a competitor tRNA) does exist simultaneously in the translation system (Fig. 6 b).This hypothesis is supported by the following fact. In starfish mitochondria, AUA is an isoleucine codon, which differs from other known animal mitochondria in which AUA is used as a methionine codon (2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar). We found that isoleucine and methionine tRNAs of starfish have unmodified GAU and CAU anticodons, respectively (data not shown). Our observation that the wobble nucleoside of the sole mammalian mt methionine tRNA is 5-formylcytidine led us to propose the possibility that this modification allows decoding of AUA in addition to AUG (5Moriya J. Yokogawa T. Wakita K. Ueda T. Nishikawa K. Crain P.F. Hashizume T. Pomerantz S.C. McCloskey J.A. Kawai G. Hayashi N. Yokoyama S. Watanabe K. Biochemistry. 1994; 33: 2234-2239Crossref PubMed Scopus (85) Google Scholar). These codon-anticodon relationships for methionine and isoleucine in mammalian and starfish mitochondria support the above interpretation on the decoding properties of unmodified guanosine at the wobble position. Since the unmodified CAU anticodon of methionine tRNA has no capability of decoding AUA (1Osawa S. Evolution of the Genetic Code. Oxford University Press, Oxford, UK1995Google Scholar, 2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar) due to the lack of modification at the wobble position, the tRNAMet does not compete with tRNAIle in translating the AUA codon, so that tRNAIle possessing the GAU anticodon can translate not only AUC and AUU, but also AUA. Thus it can be concluded that guanosine at the wobble position decodes C, U, and A.In ascidian mitochondria, AGR codons are specific for glycine, while AGY codons are translated as serine (11Yokobori S. Ueda T. Watanabe K. J. Mol. Evol. 1993; 36: 1-8Crossref PubMed Scopus (41) Google Scholar). The corresponding mt tRNAs were isolated and sequenced; tRNAGCUSer was found to have unmodified G at the wobble position, whereas tRNAGly specific for AGR has cmnm5U at the same position (30Kondo A. Yokobori S. Ueda T. Watanabe K. Nucleic Acids Symp. Ser. 1996; 35: 279-280Google Scholar). In this case, tRNAGly may compete with tRNAGCUSer, preventing AGR codons from being mistranslated as Ser. In mammalian mitochondria, only AGU and AGC codons are translated by tRNAGCUSerpossessing the anticodon GCU, and there are no tRNAs that translate the AGA and AGG codons (1Osawa S. Evolution of the Genetic Code. Oxford University Press, Oxford, UK1995Google Scholar, 2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar). Although it is apparently in contradiction to the above-mentioned codon-anticodon rule, AGG and AGA are utilized as termination codons in mammalian mitochondria (10Anderson S. Bankier A.T. Barrell B.G. de Bruijn M.H.L. Coulson A.R. Drouin J. Eperon I.C. Nierlich D.P. Roe B.A. Sanger F. Schreier P.H. Smith A.J.H. Staden R. Young I.G. Nature. 1981; 290: 457-465Crossref PubMed Scopus (7535) Google Scholar, 31Osawa S. Ohama T. Jukes T.H. Watanabe K. J. Mol. Evol. 1989; 29: 202-207Crossref PubMed Scopus (35) Google Scholar). We propose here that a release factor (1Osawa S. Evolution of the Genetic Code. Oxford University Press, Oxford, UK1995Google Scholar, 2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar) recognizing these codons plays a role similar to that of the competitor tRNA (Fig. 6 c). Thus, only AGY codons remain for assignment to serine.The present finding that m7G is the wobble nucleotide in starfish mt tRNAGCUSer suggests that methylation of G at N7 expands codon-anticodon interaction to permit base pairing with G, in addition to A, C, and U, at the third position of the codon (Fig. 6 d). Although the influence of the altered charge distribution (resulting form N7 methylation) on the base pairing of m7G with G is unknown, we are now carrying out experiments using an in vitro translation system to confirm the decoding properties of tRNA possessing m7G at the wobble position, and also to verify the ability of m7G to base pair with all four nucleotides with respect to the structural factors. We report here the first known occurrence of m7G at the wobble position of a tRNA. Even since we first discovered that in starfish mitochondria AGN codons seem to be translated as serine by the tRNAGCUSer from the sole corresponding gene located on the mt genome (6Himeno H. Masaki H. Kawai T. Ohta T. Kumagai I. Miura K.-I. Watanabe K. Gene (Amst.). 1987; 56: 219-230Crossref PubMed Scopus (77) Google Scholar), the question as to how tRNAGCUSer possessing a GCU anticodon decodes all four AGN codons has attracted our interest, especially with regard to the decoding mechanism of mt tRNAs toward nonuniversal codons. This long-pending puzzle has now been solved by the finding that a modified residue (m7G) exists at the wobble position of tRNAGCUSer, which is probably responsible for decoding AGN codons. This solution seems to be the simplest one among the speculations so far postulated (see Introduction) (1Osawa S. Evolution of the Genetic Code. Oxford University Press, Oxford, UK1995Google Scholar, 2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar,17Watanabe K. Osawa S. Soll D. RajBhandary U.L. tRNA Structure, Biosynthesis and Function. ASM Press, Washington, D. C.1995: 225-250Google Scholar). We thus consider that the occurrence of m7G at the wobble position, which has emerged uniquely in echinoderm mitochondria, leads to non-canonical base pairing. This, together with the results concerning codon-anticodon relationships obtained so far for various animal mitochondria (3Heckman J.E. Sarnoff J. Alzner-DeWeerd B. Yin S. RajBhandary U.L. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3159-3163Crossref PubMed Scopus (204) Google Scholar, 4Martin R.P. Sibler A.P. Gehrke C.W. Kuo K. Edmonds C.G. McCloskey J.A. Dirheimer G. Biochemistry. 1990; 29: 956-959Crossref PubMed Scopus (34) Google Scholar, 29Barrell B.G. Anderson S. Bankier A.T. de Bruijn M.H. Chen E. Coulson A. Drouin J. Eperon I.C. Nierlich D.P. Roe B.A. Sanger F. Schreier P.H. Smith A.J. Staden R. Young I.G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3164-3166Crossref PubMed Scopus (229) Google Scholar), enables us to draw up a set of general rules for codon-anticodon interaction in mitochondria. These are summarized in Fig. 6. In conventional mitochondrial wobble rules so far elucidated (3Heckman J.E. Sarnoff J. Alzner-DeWeerd B. Yin S. RajBhandary U.L. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3159-3163Crossref PubMed Scopus (204) Google Scholar, 4Martin R.P. Sibler A.P. Gehrke C.W. Kuo K. Edmonds C.G. McCloskey J.A. Dirheimer G. Biochemistry. 1990; 29: 956-959Crossref PubMed Scopus (34) Google Scholar,29Barrell B.G. Anderson S. Bankier A.T. de Bruijn M.H. Chen E. Coulson A. Drouin J. Eperon I.C. Nierlich D.P. Roe B.A. Sanger F. Schreier P.H. Smith A.J. Staden R. Young I.G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3164-3166Crossref PubMed Scopus (229) Google Scholar), tRNAs with unmodified guanosine in the first anticodon position are considered to translate only codons terminating in a pyrimidine at the third position, while anticodons with modified uridine at the same position translate codons having a purine at the third position (Fig. 6 a). In Drosophila mitochondria, alignment of mt genes suggests that AGA is a serine codon, whereas AGG is an unassigned codon (2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar), because AGG has never appeared in Drosophila mt genomes. We have isolated Drosophila melanogastertRNAGCUSer, which presumably recognize codons AGC, AGU, and AGA (15Okimoto R. Macfarlane J.L. Clary D.O. Wolstenholme D.R. Genetics. 1992; 130: 471-498Crossref PubMed Google Scholar), and found that unmodified guanosine is the wobble nucleotide (unpublished observation). Based on the mt genomic sequence, it is unlikely that any tRNA competing with tRNAGCUSer with regard to the translation of codon AGA exists. Thus, unmodified guanosine is probably able to base pair with adenosine at the codon third position, unless a tRNA that efficiently decodes this codon (a competitor tRNA) does exist simultaneously in the translation system (Fig. 6 b). This hypothesis is supported by the following fact. In starfish mitochondria, AUA is an isoleucine codon, which differs from other known animal mitochondria in which AUA is used as a methionine codon (2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar). We found that isoleucine and methionine tRNAs of starfish have unmodified GAU and CAU anticodons, respectively (data not shown). Our observation that the wobble nucleoside of the sole mammalian mt methionine tRNA is 5-formylcytidine led us to propose the possibility that this modification allows decoding of AUA in addition to AUG (5Moriya J. Yokogawa T. Wakita K. Ueda T. Nishikawa K. Crain P.F. Hashizume T. Pomerantz S.C. McCloskey J.A. Kawai G. Hayashi N. Yokoyama S. Watanabe K. Biochemistry. 1994; 33: 2234-2239Crossref PubMed Scopus (85) Google Scholar). These codon-anticodon relationships for methionine and isoleucine in mammalian and starfish mitochondria support the above interpretation on the decoding properties of unmodified guanosine at the wobble position. Since the unmodified CAU anticodon of methionine tRNA has no capability of decoding AUA (1Osawa S. Evolution of the Genetic Code. Oxford University Press, Oxford, UK1995Google Scholar, 2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar) due to the lack of modification at the wobble position, the tRNAMet does not compete with tRNAIle in translating the AUA codon, so that tRNAIle possessing the GAU anticodon can translate not only AUC and AUU, but also AUA. Thus it can be concluded that guanosine at the wobble position decodes C, U, and A. In ascidian mitochondria, AGR codons are specific for glycine, while AGY codons are translated as serine (11Yokobori S. Ueda T. Watanabe K. J. Mol. Evol. 1993; 36: 1-8Crossref PubMed Scopus (41) Google Scholar). The corresponding mt tRNAs were isolated and sequenced; tRNAGCUSer was found to have unmodified G at the wobble position, whereas tRNAGly specific for AGR has cmnm5U at the same position (30Kondo A. Yokobori S. Ueda T. Watanabe K. Nucleic Acids Symp. Ser. 1996; 35: 279-280Google Scholar). In this case, tRNAGly may compete with tRNAGCUSer, preventing AGR codons from being mistranslated as Ser. In mammalian mitochondria, only AGU and AGC codons are translated by tRNAGCUSerpossessing the anticodon GCU, and there are no tRNAs that translate the AGA and AGG codons (1Osawa S. Evolution of the Genetic Code. Oxford University Press, Oxford, UK1995Google Scholar, 2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar). Although it is apparently in contradiction to the above-mentioned codon-anticodon rule, AGG and AGA are utilized as termination codons in mammalian mitochondria (10Anderson S. Bankier A.T. Barrell B.G. de Bruijn M.H.L. Coulson A.R. Drouin J. Eperon I.C. Nierlich D.P. Roe B.A. Sanger F. Schreier P.H. Smith A.J.H. Staden R. Young I.G. Nature. 1981; 290: 457-465Crossref PubMed Scopus (7535) Google Scholar, 31Osawa S. Ohama T. Jukes T.H. Watanabe K. J. Mol. Evol. 1989; 29: 202-207Crossref PubMed Scopus (35) Google Scholar). We propose here that a release factor (1Osawa S. Evolution of the Genetic Code. Oxford University Press, Oxford, UK1995Google Scholar, 2Osawa S. Jukes T.H. Watanabe K. Muto A. Microbiol. Rev. 1992; 56: 229-264Crossref PubMed Google Scholar) recognizing these codons plays a role similar to that of the competitor tRNA (Fig. 6 c). Thus, only AGY codons remain for assignment to serine. The present finding that m7G is the wobble nucleotide in starfish mt tRNAGCUSer suggests that methylation of G at N7 expands codon-anticodon interaction to permit base pairing with G, in addition to A, C, and U, at the third position of the codon (Fig. 6 d). Although the influence of the altered charge distribution (resulting form N7 methylation) on the base pairing of m7G with G is unknown, we are now carrying out experiments using an in vitro translation system to confirm the decoding properties of tRNA possessing m7G at the wobble position, and also to verify the ability of m7G to base pair with all four nucleotides with respect to the structural factors. The authors are grateful to Kozo Tomita, the University of Tokyo, Dr. S. Yokobori, Tokyo University of Pharmacy and Life Science, and Dr. Y. Watanabe, Dalhousie University, for helpful discussions. We also thank Dr. S. Osawa, Biohistory Research Institute, for encouragement." @default.
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W1979201731 title "A Novel Wobble Rule Found in Starfish Mitochondria" @default.
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