Figure 4.

Effect of acetylation of Lys residues on the Leu activation and aminoacylation activities of EcLeuRS. A, sequence alignment of LeuRSs from various species in regions homologous to Lys⁶¹⁹, Lys⁶²⁴, and Lys⁸⁰⁹ in EcLeuRS. Ec, E. coli; Aa, Aquifex aeolicus; Bs, B. subtilis; Sco, Streptomyces coelicolor; Hs, H. sapiens; Ph, Pyrococcus horikoshii; mt, mitochondrial; ct, cytoplasmic. B, Western blotting confirming the incorporation of AcK in EcLeuRS-K619^Ac, EcLeuRS-K624^Ac, and EcLeuRS-K809^Ac. C, Leu activation of EcLeuRS-K619^Ac, EcLeuRS-K624^Ac, and EcLeuRS-K809^Ac. D, aminoacylation of EcLeuRS-K619^Ac, EcLeuRS-K624^Ac, and EcLeuRS-K809^Ac resembling that of the K-Q mutants. E, closer view of the orientation of Lys⁶¹⁹ and Lys⁶²⁴ relative to the conserved HIGH and KMSK motifs (HMGH and KMSK in EcLeuRS, depicted in dark blue; PDB code 4ARC). F, closer view of the interaction between EcLeuRS Lys⁸⁰⁹ and EctRNA^Leu U47I (PDB code 4ARC). The results are the averages and standard deviations from three independent experiments, and all Western blots were repeated.