A UGU sequence in the anticodon loop is a minimum requirement for recognition by Escherichia coli tRNA-guanine transglycosylase.
Escherichia coli tRNA-guanine transglycosylase is an enzyme which catalyzes replacement of guanine (G34) of tRNA(Asp), tRNA(Asn), tRNA(His) and tRNA(Tyr) by free guanine or free preQ1 base by a base exchange reaction in the biosynthesis of queuosine (Q) (Okada, N., and Nishimura, S. (1979) J. Biol. Chem. 254, 3061-3066). The gene encoding for this enzyme was amplified from the E. coli genome by polymerase chain reaction and inserted into an overexpression vector, pJLA503. The enzyme was overexpressed by heat induction in E. coli transformed by this recombinant plasmid and purified to homogeneity by two column chromatographies. The sequence requirement in tRNA for recognition by this enzyme was investigated using minihelices corresponding to the anti-codon arm of E. coli tRNA(His). Two uridine residues (U33, U35) were found to be prerequisite for such recognition by this enzyme. Position 32 required pyrimidines, because the enzyme activity toward the minihelices was markedly reduced or entirely lost when this residue was replaced by purines or was deleted. Adenosine at position 37 and the G30-C40 base pair were not essential despite their conservation. Our results suggest that the enzyme recognizes the U33-G34-U35 sequence in the anti-codon loop and not the tertiary structure of tRNA itself.
Nakanishi
S
S
Department of Chemistry and Biotechnology, Faculty of Engineering, University of Tokyo, Japan.
Ueda
T
T
Hori
H
H
Yamazaki
N
N
Okada
N
N
Watanabe
K
K
D000926:Anticodon; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010430:Pentosyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012329:RNA, Bacterial; D012343:RNA, Transfer / Q000235:genetics; D013379:Substrate Specificity
D000926:Anticodon; D012329:RNA, Bacterial; D012343:RNA, Transfer; D010430:Pentosyltransferases; C020837:queuine tRNA-ribosyltransferase