Overproduction of tyrosyl-tRNA synthetase is toxic to Escherichia coli: a genetic analysis.
The tyrS genes from Escherichia coli and Bacillus stearothermophilus were toxic to E. coli when they were carried by plasmids with very high copy numbers (pEMBL8 and pEMBL9). We quantified this effect by comparing the efficiencies of plating of E. coli derivatives harboring recombinant plasmids in various experimental conditions. The toxicity was apparent at both 30 and 37 degrees C. It increased with the growth temperature, the strength of the tyrS promoter, and the copy number of the plasmidic vector. Two- to threefold enhancement of tyrS expression raised the toxicity 300-fold. Point mutations in tyrS that prevent interaction between its product, tyrosyl-tRNA synthetase, and tRNA(Tyr) but do not alter the rate of formation of tyrosyl-adenylate abolished the toxicity. Thus, the toxic effect was due to high cellular levels of synthetase activity. At 30 degrees C, the cellular concentration of tyrosyl-tRNA synthetase reached 55% of that of soluble proteins and led to decreased beta-galactosidase stability. We discuss possible causes of this toxic effect and describe its applications to the study of the recognition and interaction between the synthetase and tRNA(Tyr).
Bedouelle
H
H
Unité de Biochimie Cellulaire, Institut Pasteur, Paris, France.
Guez
V
V
Vidal-Cros
A
A
Hermann
M
M
D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D003090:Coliphages / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D010957:Plasmids; D015183:Restriction Mapping; D013696:Temperature; D014448:Tyrosine-tRNA Ligase / Q000096:biosynthesis / Q000235:genetics*; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors
D007544:Isopropyl Thiogalactoside; D001616:beta-Galactosidase; D000604:Amino Acyl-tRNA Synthetases; D014448:Tyrosine-tRNA Ligase