.::) 1992 Oxford University Press

Nucleic Acids Research, Vol. 20, No. 15 4089

Nucleotide sequence of tRNATh,i of Escherichia coli and of the gene (thrV that encodes it Yuriko Komine and Hachiro Inokuchi* Department of Biophysics, Faculty of Science, Kyoto University, Kyoto 606-01, Japan Submitted July 9, 1992 We purified a tRNA that hybridized to a synthetic DNA probe (5'-CTGGGGACCCCACCCCTACCAAGGGTGCGC) which was complementary to part of the reported sequence of the gene (thrV) that encodes tRNAThrl (1) from Escherichia coli K-12 W3110. We determined the nucleotide sequence of the tRNA, including modified bases, by the method described in (2) (Figure 1). However, our RNA sequence was inconsistent with the DNA sequence of thrV at two positions (nucleotides A38 and A44 in the RNA). The thrV gene is located in the distal region of the rinD operon (1), which is included in the genome of phage clones 531 and 532 of Kohara's library (3, 4). We cloned the thrV region and determined its sequence. Four nucleotides (two of them were beyond the coding region) were different from those in the previously reported sequence and the entire sequence of the coding region was consistent with the RNA sequence in Figure 1. We concluded, therefore, that the tRNA that we purified and sequenced is indeed tRNAThrI and that the sequence of thrV, which encodes it, is different from the previously reported sequence, at least in W3110. The sequence of tRNAThr, is very similar to that of tRNAThr3, an isoacceptor with the same anticodon (GGU) (5, 6). Moreover, it has exactly the same modified bases at the same positions as those of tRNAThr3. In our previous report, we summarized DNA sequences for all species of tRNA (46 species) of E.coli (4). However, the sequence for tRNAThrI should be corrected and replaced by the present sequence. Kleina et al. reported that a synthetic amber suppressor based on the thrV sequence functioned very poorly, although the other suppressor based on the sequence of another tRNAThr can function efficiently (7). The poor performance of the former suppressor might be due to the inclusion of incorrect bases (G38 and G44).

DDBJ accession no. D12500 4. Komine,Y., Adachi,T., Inokuchi,H. and Ozeki,H. (1990) J. Mol. Bio. 212, 579-598. 5. Clarke,L. and Carbon,J. (1974) J. Biol. Chem. 249, 6874-6885. 6. An,G. and Friesen,J.D. (1980) Gene 12, 33-39. 7. Kleina,L.G., Maddon,J.-M., Normanly,J., Abelson,J. and Miller,J.H. (1990) J. Mol. Biol. 213, 705-717.

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ACKNOWLEDGMENT This work was supported by a Grant-in-Aid for Special Research Projects from the Ministry of Education, Science and Culture of Japan. REFERENCES 1. Duester,D.L. and Holmes,W.M. (1980) Nucleic Acids Res. 8, 3793 -3807. 2. Kuchino,Y., Hanyu,N. and Nishimura,S. (1987) Methods Enzymol. 155, 379-396. 3. Kohara,Y., Akiyama,K. and Isono,K. (1987) Cell 50, 495-508.

*

To whom correspondence should be addressed

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G G U Figure 1. Nucleotide sequence of tRNAThTI from Escherichia coli, arranged in a clover-leaf form. Modified bases are: D, dihydrouridine; F, pseudouridine; T, ribothymidine; mt6A, N-[(9- 13-D-ribofuranosylpurin-6-yl)-N-methylcarbamoyl]-threonine; m7G, 7-methylguanosine. The nucleotides that are different from those in the sequence encoded by thrV in reference (1) are indicated in bold letters. The base substitutions in tRNAThr3 are shown in small letters.