Modified nucleosides in the first positions of the anticodons of tRNA(4)(Leu) and tRNA(5)(Leu) from Escherichia coli

Minor leucine tRNA species, tRNA(4)(Leu) and tRNA(5)(Leu), from Escherichia coli B have been reported to recognize leucine codons UUA and UUG [Goldman , E., Holmes, W. M., and Hatfield, G. W. (1979) J. Mel. Biol. 129, 567-585] . In the present study, these two tRNA(Leu) species were purified from E. c oli A19, and the nucleotide sequences were determined by a post-labeling me thod. tRNA(5)(Leu) was found to correspond to the tRNA gene reported as su degrees 6 tRNA [Yoshimura, M., Inokuchi, H., and Ozeki, H. (1984) J. Mel. B iol. 177, 627-644]. The first letter of the anticodon was identified to be 2'-O-methylcytidine (Cm). tRNA(4)(Leu) was identified as the minor leucine tRNA that has been sequenced previously (tRNA(UUR)(Leu)) [Yamaizumi, Z., Ku chino, Y., Harada, F., Nishimura, S., and McCloskey, J. A. (1980) J. Biol. Chem. 255, 2220-2225]. There was an unidentified modified nucleoside (N*) i n the first position of the anticodon of tRNA(4)(Leu). Nucleoside N* was is olated to homogeneity (1 A(260) Unit). By H-1 NMR spectroscopy, nucleoside N* was found to be a 2'-O-methyluridine derivative with a substituent havin g a -CH2NH2+CH2COO- moiety in position 5 of the uracil ring. On the basis o f these NMR analyses together with mass spectrometry, the chemical structur e of nucleoside N* was determined as 5-carboxymethylaminomethyl-2'-O-methyl uridine (cmnm(5)Um). Nucleoside N* was thus found to be a novel type of nat urally occurring modified uridine. Because of the conformational rigidity o f Cm and cmnm5Um in the first position of the anticodon, these tRNA(Leu) sp ecies recognize the leucine codons UUA and UUG correctly, but never recogni ze the phenylalanine codons UUU and UUC.