The modified wobble base inosine in yeast tRNAIle is a positive determinant for aminoacylation by isoleucyl-tRNA synthetase.
Earlier work by two independent groups has established the fact that anticodons GAU and LAU of Escherichia coli tRNAIle isoacceptors play a critical role in the tRNA identity. Yeast possesses two isoleucine transfer RNAs, a major one with anticodon IAU and a minor one with anticodon PsiAPsi which are derived from the post-transcriptional modification of AAU and UAU gene sequences, respectively. We present direct evidence which reveals that inosine is a positive determinant for yeast isoleucyl-tRNA synthetase. We also show that yeast tRNAMet with guanosine at the wobble position becomes aminoacylated with isoleucine while methionine acceptance is lost. As inosine and guanosine share the 6-keto and the N-1 hydrogen groups, this suggests that these hydrogen donor and acceptor groups are determinants for isoleucine specificity. The role of the minor tRNAIle anticodon pseudouridines in tRNA isoleucylation could not be tested directly but was deduced from a 40-fold decrease in the activity of the unmodified transcript. The presence of the NHCO structure in guanosine, inosine, pseudouridine, and lysidine suggests a unifying model of wobble base recognition by the yeast and E. coli isoleucyl-tRNA synthetase. In contrast to lysidine which switches the identity of the tRNA from methionine to isoleucine [Muramatsu, T., Nishikawa, K., Nemoto, F., Kuchino, Y., Nishimura, S., Miyazawa, T., & Yokoyama, S. (1988) Nature 336, 179-181], pseudouridine-34 does not modify the specificity of the yeast minor tRNAIle since U-34 is a strong negative determinant for yeast MetRS. Therefore, the major role of Psi-34 (in combination with Psi-36 or not) is likely in isoleucine AUA codon specificity and translational fidelity.
Senger
B
B
Institut de Biologie Moléculaire et Cellulaire, UPR 9002 15, Rue René Descartes, F-67084 Strasbourg Cedex, France.
Auxilien
S
S
Englisch
U
U
Cramer
F
F
Fasiolo
F
F
D000215:Acylation; D000926:Anticodon; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D007288:Inosine / Q000737:chemistry* / Q000378:metabolism; D007533:Isoleucine-tRNA Ligase / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011560:Pseudouridine / Q000737:chemistry / Q000378:metabolism; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D012331:RNA, Fungal / Q000737:chemistry / Q000378:metabolism; D012355:RNA, Transfer, Ile / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000926:Anticodon; D012329:RNA, Bacterial; D012331:RNA, Fungal; D012355:RNA, Transfer, Ile; D011560:Pseudouridine; D007288:Inosine; D007533:Isoleucine-tRNA Ligase