title: Role of residue Glu152 in the discrimination between transfer RNAs by tyrosyl-tRNA synthetase from Bacillus stearothermophilus.
abstract: Residue Glu152 of tyrosyl-tRNA synthetase (TyrTS) from Bacillus stearothermophilus is close to phosphate groups 73 and 74 of tRNATyr in the structural model of their complex. TyrTS(E152A), a mutant synthetase carrying the change of Glu152 to Ala, was toxic when overproduced in Escherichia coli. The toxicity strongly increased with the growth temperature. It was measured by the ratios of the efficiencies with which the producing cells plated in induced or repressed conditions and at 30 degrees C or 37 degrees C. TyrTS(E152Q), TyrTS(E152D) and the wild-type synthetase were not toxic in conditions where TyrTS(E152A) was toxic. The toxicity of TyrTS(E152A) was abolished by additional mutations of the synthetase that prevent the binding of tRNATyr but not by a mutation that prevents the formation of Tyr-AMP. Because TyrTS(E152A) was active for the aminoacylation of tRNATyr, its toxicity could only be due to faulty interactions with non-cognate tRNAs, either their non-productive binding or their mischarging with tyrosine. TyrTS(E152A) and TyrTS(E152Q) mischarged tRNAPhe and tRNAVal in vitro with tyrosine unlike TyrTS(E152D) or the wild-type enzyme. Thus, several features of the side-chain in position 152 of TyrTS, including its negative charge, are important for the rejection of non-cognate tRNAs. TyrTS(E152A), TyrTS(E152D) and TyrTS(E152Q) had similar steady-state kinetics parameters for the charging of tRNATyr with tyrosine in vitro, with kcat/KM ratios improved 2.5 times relative to the wild-type synthetase. We conclude that the side-chain of residue Glu152 weakens the binding of TyrTS to tRNATyr and prevents its interaction with non-cognate tRNAs.
authors.affiliation: Unité de Biochimie Cellulaire (CNRS URA 1129) Institut Pasteur, Paris, France.
authors.forename: A
authors.initials: A
authors.lastname: Vidal-Cros
authors.forename: H
authors.initials: H
authors.lastname: Bedouelle
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism; D004563:Electrochemistry; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D005973:Glutamine / Q000378:metabolism*; D007700:Kinetics; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D012365:RNA, Transfer, Tyr / Q000378:metabolism*; D015183:Restriction Mapping; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D012365:RNA, Transfer, Tyr; D005973:Glutamine; D000249:Adenosine Monophosphate; D014443:Tyrosine; D014448:Tyrosine-tRNA Ligase