.=; 1991 Oxford University Press
Nucleic Acids Research, Vol. 19, No. 8 1941
Nucleotide sequence and functional characterization of a mitochondrial tRNATrP from Tetrahymena therrmophila Armin Hekele and Hildburg Beier Institut fOr Biochemie, Bayerische Julius-Maximilians-Universitat, Rontgenring 11, D-8700 Wurzburg, FRG EMBL accession no. X58116
Submitted February 15, 1991 The use of the termination codon UGA as a tryptophan codon in mitochondria appears to be one of the most common deviations from the standard genetic code. This change in the mitochondrial genome is accomodated by the presence of an anticodon U*CA in mitochondrial (mt) tRNATrP (1). Recently a tRNA gene with a TCA anticodon has been identified in the mt genome of the ciliates Paramecium tetraurelia (2) and Tetrahymena pyriformis (3). Here we describe a mt tRNATrP with the anticodon U*CA from Tetrahymena thermophila. Total tRNA and aminoacyl-tRNA synthetase were isolated from the cells of T. thermophila as described previously (4). Fractionation of tRNAs by BD-cellulose column chromatography, purification of tRNAs by polyacrylamide gel electrophoresis and sequencing of tRNA by post-labelling techniques was carried out according to Beier et al. (5). UGA suppressor activity of T. thermophila column or gel fractions iwas assayed in a reticulocyte lysate programmed with globin mRNA (6). Only one tRNA species capable of suppressing the UGA termination codon in ,B-globin mRNA was identified in fractions of total Tetrahymena tRNA. This tRNA is charged with tryptophan by a crude Tetrahymena aminoacyl-tRNA synthetase preparation. The nucleotide sequence of the tRNAThP is shown in Fig. 1. It exhibits a high degree of homology with the known sequence of the mt tRNATrP gene from T. pyriformis (3), differing only in two nucleotides at positions 16 and 69. Consequently, we conclude that we have identified the corresponding T. thermophila tRNA with a U*CA anticodon. The modified uridine (U*) in position 34 is very likely 5[[(carboxymethyl)amino]methyl]uridine (cmnm5U) as indicated by its thin-layer chromatographic mobility in two solvent systems (4). U55 is only partially modified to pseudouridine. A* is probably N6-isopentenyladenosine (i6A) or 2-methylthio-N6-isopentenyladenosine
REFERENCES 1. Sprinzl,M., Hartmann,T., Weber,J., Blank,J. and Zeidler,R. (1989) Nucl. Acids Res. 17, rl-r172. 2. Suyama,Y. (1985) Nucl. Acids Res. 13, 3273-3284. 3. Seilhamer,J.J. and Cummings,D.J. (1982) Mol. Gen. Genet. 187, 236-239. 4. Kuchino,Y., Hanyu,N., Tashiro,F. and Nishimura,S. (1985) Proc. Natl. Acad. Sci. USA 82, 4758-4762. 5. Beier,H., Barciszewska,M., Krupp,G., Mitnacht,R. and Gross,H.J. (1984) EMBO J. 3, 351-356. 6. Hanyu,N., Kuchino,Y., Nishimura,S. and Beier,H. (1986) EMBO J. 5, 1307-1311.
c
Pc-c c- U c-c G-C
UAAC
UUU3GA
A-U A-U U CGCUC C
I : : I I C
C AA C GDADa
la
UA G )
G
A-U C-C C-C C-- '1' U-A c A U A U' C A
Figure 1. Cloverleaf structure of mitochondrial tryptophan tRNA from Tetrahymena thermophila.
(ms2i6A).
The Tetrahymena mt tRNATrP (U*CA) functions in cytoplasmic protein synthesis as demonstrated in vitro by the efficient suppression of the UGA termination codon of (-globin mRNA in a reticulocyte lysate (Fig. 2). However, the suppression is strictly dependent on the simultaneous presence of aminoacyltRNA synthetase from Tetrahymena cells.
ACKNOWLEDGEMENTS We thank Dr. H.-M. Seyfert (Giessen) for a culture of Tetrahymena thermophila cells and Karin Zerfass for a gift of Tetrahymena aminoacyl-tRNA synthetase. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to H.B.
a-
a/W a
b
c
d
Figure 2. Translation of globin mRNA in a reticulocyte lysate in the presence of 50 ng4Al Tetrahymena mt tRNATrlP (a), yeast UGA (b) and yeast UAA suppressor tRNA (c) or in the absence of added tRNA (d). The readthrough products of a- and (-globin mRNA are indicated ca' and (3', respectively.
Nucleic Acids Research, Vol. 19, No. 8 1941
Nucleotide sequence and functional characterization of a mitochondrial tRNATrP from Tetrahymena therrmophila Armin Hekele and Hildburg Beier Institut fOr Biochemie, Bayerische Julius-Maximilians-Universitat, Rontgenring 11, D-8700 Wurzburg, FRG EMBL accession no. X58116
Submitted February 15, 1991 The use of the termination codon UGA as a tryptophan codon in mitochondria appears to be one of the most common deviations from the standard genetic code. This change in the mitochondrial genome is accomodated by the presence of an anticodon U*CA in mitochondrial (mt) tRNATrP (1). Recently a tRNA gene with a TCA anticodon has been identified in the mt genome of the ciliates Paramecium tetraurelia (2) and Tetrahymena pyriformis (3). Here we describe a mt tRNATrP with the anticodon U*CA from Tetrahymena thermophila. Total tRNA and aminoacyl-tRNA synthetase were isolated from the cells of T. thermophila as described previously (4). Fractionation of tRNAs by BD-cellulose column chromatography, purification of tRNAs by polyacrylamide gel electrophoresis and sequencing of tRNA by post-labelling techniques was carried out according to Beier et al. (5). UGA suppressor activity of T. thermophila column or gel fractions iwas assayed in a reticulocyte lysate programmed with globin mRNA (6). Only one tRNA species capable of suppressing the UGA termination codon in ,B-globin mRNA was identified in fractions of total Tetrahymena tRNA. This tRNA is charged with tryptophan by a crude Tetrahymena aminoacyl-tRNA synthetase preparation. The nucleotide sequence of the tRNAThP is shown in Fig. 1. It exhibits a high degree of homology with the known sequence of the mt tRNATrP gene from T. pyriformis (3), differing only in two nucleotides at positions 16 and 69. Consequently, we conclude that we have identified the corresponding T. thermophila tRNA with a U*CA anticodon. The modified uridine (U*) in position 34 is very likely 5[[(carboxymethyl)amino]methyl]uridine (cmnm5U) as indicated by its thin-layer chromatographic mobility in two solvent systems (4). U55 is only partially modified to pseudouridine. A* is probably N6-isopentenyladenosine (i6A) or 2-methylthio-N6-isopentenyladenosine
REFERENCES 1. Sprinzl,M., Hartmann,T., Weber,J., Blank,J. and Zeidler,R. (1989) Nucl. Acids Res. 17, rl-r172. 2. Suyama,Y. (1985) Nucl. Acids Res. 13, 3273-3284. 3. Seilhamer,J.J. and Cummings,D.J. (1982) Mol. Gen. Genet. 187, 236-239. 4. Kuchino,Y., Hanyu,N., Tashiro,F. and Nishimura,S. (1985) Proc. Natl. Acad. Sci. USA 82, 4758-4762. 5. Beier,H., Barciszewska,M., Krupp,G., Mitnacht,R. and Gross,H.J. (1984) EMBO J. 3, 351-356. 6. Hanyu,N., Kuchino,Y., Nishimura,S. and Beier,H. (1986) EMBO J. 5, 1307-1311.
c
Pc-c c- U c-c G-C
UAAC
UUU3GA
A-U A-U U CGCUC C
I : : I I C
C AA C GDADa
la
UA G )
G
A-U C-C C-C C-- '1' U-A c A U A U' C A
Figure 1. Cloverleaf structure of mitochondrial tryptophan tRNA from Tetrahymena thermophila.
(ms2i6A).
The Tetrahymena mt tRNATrP (U*CA) functions in cytoplasmic protein synthesis as demonstrated in vitro by the efficient suppression of the UGA termination codon of (-globin mRNA in a reticulocyte lysate (Fig. 2). However, the suppression is strictly dependent on the simultaneous presence of aminoacyltRNA synthetase from Tetrahymena cells.
ACKNOWLEDGEMENTS We thank Dr. H.-M. Seyfert (Giessen) for a culture of Tetrahymena thermophila cells and Karin Zerfass for a gift of Tetrahymena aminoacyl-tRNA synthetase. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to H.B.
a-
a/W a
b
c
d
Figure 2. Translation of globin mRNA in a reticulocyte lysate in the presence of 50 ng4Al Tetrahymena mt tRNATrlP (a), yeast UGA (b) and yeast UAA suppressor tRNA (c) or in the absence of added tRNA (d). The readthrough products of a- and (-globin mRNA are indicated ca' and (3', respectively.
