Modulation of the suppression efficiency and amino acid identity of an artificial yeast amber isoleucine transfer RNA in Escherichia coli by a G-U pair in the anticodon stem.
The artificial amber suppressor corresponding to the major isoleucine tRNA from yeast (pVBt5), when expressed in E. coli, is a poor suppressor of the amber mutation lacIam181-Z. By analysing mutant forms, we could show that this was due to the presence of a U30-G40 wobble pair in the anticodon stem of the yeast tRNA and not to the level of the heterologously expressed tRNA. Efficient suppressors were obtained by restoring a normal U30-A40 or G30-C40 Watson-Crick pair. In vivo the mutant forms are exclusively charged by the bacterial lysyl-tRNA synthetase (LysRS), whereas the original yeast amber tRNA is charged at a low level by E. coli glutaminyl-tRNA synthetase (GlnRS) and LysRS. The inversion of the U30-G40 pair also induces a loss of the Gln identity. We conclude from these experiments that the U30-G40 base pair constitutes a negative determinant for LysRS interaction which operates either at the level of complex formation or at the catalytic step. As no direct contacts are seen between GlnRS and positions 30-40 of the complexed homologous tRNA, the U30-G40 pair of pVBt5 is believed to influence aminoacylation by GlnRS indirectly, probably at the level of the anticodon loop conformation by favouring an optimal apposition of the anticodon nucleotides with the protein.
Büttcher
V
V
UPR 9002 du CNRS, Institute de Biologie Moléculaire et Cellulaire, Strasbourg, France.
Senger
B
B
Schumacher
S
S
Reinbolt
J
J
Fasiolo
F
F
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D000926:Anticodon / Q000235:genetics*; D001482:Base Composition; D001483:Base Sequence; D007446:Chromosome Inversion; D018244:Chromosomes, Artificial, Yeast; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D006147:Guanine; D008250:Lysine-tRNA Ligase / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D010957:Plasmids; D012351:RNA, Transfer, Gln / Q000737:chemistry / Q000235:genetics; D012355:RNA, Transfer, Ile / Q000737:chemistry / Q000235:genetics*; D012357:RNA, Transfer, Lys / Q000737:chemistry / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013489:Suppression, Genetic; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014498:Uracil
D000926:Anticodon; D012351:RNA, Transfer, Gln; D012355:RNA, Transfer, Ile; D012357:RNA, Transfer, Lys; D014498:Uracil; D006147:Guanine; D013762:Tetrahydrofolate Dehydrogenase; D000604:Amino Acyl-tRNA Synthetases; C020659:glutaminyl-tRNA synthetase; D008250:Lysine-tRNA Ligase