A base pair at the bottom of the anticodon stem is reciprocally preferred for discrimination of cognate tRNAs by Escherichia coli lysyl- and glutaminyl-tRNA synthetases.
Although the yeast amber suppressor tRNA(Tyr) is a good candidate for a carrier of unnatural amino acids into proteins, slight misacylation with lysine was found to occur in an Escherichia coli protein synthesis system. Although it was possible to restrain the mislysylation by genetically engineering the anticodon stem region of the amber suppressor tRNA(Tyr), the mutant tRNA showing the lowest acceptance of lysine was found to accept a trace level of glutamine instead. Moreover, the glutamine-acceptance of various tRNA(Tyr) transcripts substituted at the anticodon stem region varied in reverse proportion to the lysine-acceptance, similar to a 'seesaw'. The introduction of a C31-G39 base pair at the site was most effective for decreasing the lysine-acceptance and increasing the glutamine-acceptance. When the same substitution was introduced into E.coli tRNA(Lys) transcripts, the lysine-accepting activity was decreased by 100-fold and faint acceptance of glutamine was observed. These results may support the idea that there are some structural element(s) in the anticodon stem of tRNA, which are not shared by aminoacyl-tRNA synthetases that have similar recognition sites in the anticodon, such as E.coli lysyl- and glutaminyl-tRNA synthetases.
Fukunaga
Jun-ichi
J
Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
Ohno
Satoshi
S
Nishikawa
Kazuya
K
Yokogawa
Takashi
T
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D000926:Anticodon / Q000737:chemistry; D020029:Base Pairing; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology*; D005973:Glutamine / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D008250:Lysine-tRNA Ligase / Q000378:metabolism*; D008969:Molecular Sequence Data; D012357:RNA, Transfer, Lys / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012365:RNA, Transfer, Tyr / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013489:Suppression, Genetic; D046249:Transfer RNA Aminoacylation
D000926:Anticodon; D012357:RNA, Transfer, Lys; D012365:RNA, Transfer, Tyr; D005973:Glutamine; D000604:Amino Acyl-tRNA Synthetases; C020659:glutaminyl-tRNA synthetase; D008250:Lysine-tRNA Ligase; D008239:Lysine