Modified Nucleosides in the First Positions of the Anticodons of tRNA sub(4) super(Leu) and tRNA sub(5) super(Leu) from Escherichia coli

• Published in: Biochemistry (Easton) 1999-01, Vol.38 (1), p.207-217
• Main Authors: Horie, Nobuyuki, Yamaizumi, Ziro, Kuchino, Yoshiyuki, Takai, K, Goldman, E, Miyazawa, T, Nishimura, Susumu, Yokoyama, S
• Format: Article
• Language: English
• Subjects: Escherichia coli
• ISSN: 0006-2960

Minor leucine tRNA species, tRNA sub(4) super(Leu) and tRNA sub(5) super(Leu), from Escherichia coli B have been reported to recognize leucine codons UUA and UUG. In the present study, these two tRNA super(Leu) species were purified from E. coli A19, and the nucleotide sequences were determined by a post-labeling method. tRNA sub(5) super(Leu) was found to correspond to the tRNA gene reported as su degree 6 tRNA. The first letter of the anticodon was identified to be 2'-O-methylcytidine (Cm). tRNA sub(4) super(Leu) was identified as the minor leucine tRNA that has been sequenced previously (tRNA sub(U) super(L) sub(U) super(e) sub(R) super(u)). There was an unidentified modified nucleoside (N*) in the first position of the anticodon of tRNA sub(4) super(Leu). Nucleoside N* was isolated to homogeneity (1 A sub(260) unit). By super(1)H NMR spectroscopy, nucleoside N* was found to be a 2'-O-methyluridine derivative with a substituent having a -CH sub(2)NH sub(2) super(+)CH sub(2)COO super(-) moiety in position 5 of the uracil ring. On the basis of these NMR analyses together with mass spectrometry, the chemical structure of nucleoside N* was determined as 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm super(5)Um). Nucleoside N* was thus found to be a novel type of naturally occurring modified uridine. Because of the conformational rigidity of Cm and cmnm super(5)Um in the first position of the anticodon, these tRNA super(Leu) species recognize the leucine codons UUA and UUG correctly, but never recognize the phenylalanine codons UUU and UUC.