Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

Abstract

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5')tetraphospho(5')adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

AppppA-binding protein E89 is the Escherichia coli heat shock protein ClpB

“…First, AppppA levels rise rapidly when the cell is exposed to heat (19). Second, lysU, which encodes lysyl tRNA synthetase, is itself a heat shock gene (5,6,31). Lysyl tRNA synthetase contributes significantly to AppppA production in E. coli.…”

Protein Synthesis and the Stress Response

“…These dinucleotides are synthesized by aminoacyl-tRNA synthetases during stressful conditions such as oxidative stress and heat shock [41,[64][65][66][67]. During the synthesis of aminoacyl-tRNA, aaRS produce aminoacyl-adenylate (aa-aMP) as an intermediate of the reaction (reaction 1).…”

“…This reaction can be catalyzed by several aaRS, but the most active are Phenylalanyl-tRNA synthetase (PheRS) and specially Lysyl-tRNA synthetase (LysRS) [64,69].…”

“…Probably, an important source of the increase of these molecules concentration during heat shock is that the rate of dinucleotides synthesis by LysRS increases with temperature, while the rate of Lys-tRNA Lys synthesis decreases [64]. Also, the induction at higher temperatures of a specific LysRS isoenzyme (encoded by lysU gene) that is more stable and active for dinucleotides synthesis may explain the increase in AppppN during heat shock [64].…”

“…Probably, an important source of the increase of these molecules concentration during heat shock is that the rate of dinucleotides synthesis by LysRS increases with temperature, while the rate of Lys-tRNA Lys synthesis decreases [64]. Also, the induction at higher temperatures of a specific LysRS isoenzyme (encoded by lysU gene) that is more stable and active for dinucleotides synthesis may explain the increase in AppppN during heat shock [64]. A possible explanation for the increase of these dinucleotides during oxidative stress could be the inactivation of their degradation enzyme, P1,P4-bis(5'-adenosyl) tetraphosphate pyrophosphohydrolase (ApaH), which in vitro is sensitive to cysteine oxidation [73].…”