Figure 4.
OsERS1 functions as a ligase of Glu and tRNAGlu. A, Conserved sites in the catalytic domain of OsERS1 used for site-directed mutagenesis. The figure was generated from 40 homologs of OsERS1 by WebLogo (http://weblogo.berkeley.edu). B, ATP-PPi exchange rate in the presence of increased concentrations of tRNAGlu. C, tRNAGlu aminoacylation by wild-type (WT) and mutant (M1–M3) OsERS1. D, Kinetics of tRNAGlu aminoacylation by wild-type OsERS1 and OsERS1 mutant M1 under increased tRNAGlu concentrations. Wild-type OsERS1, R2 = 0.99; OsERS1 mutant M1, R2 = 0.97. E, Kinetic constants determined by tRNAGlu charging assays. Kinetics of the OsERS1 and OsERS1 M1 to M3 aminoacylation reaction was measured using varied concentrations of tRNAGlu, and reactions were conducted for 10 min under standard conditions. Reciprocal initial velocity was plotted against the reciprocal tRNAGlu concentration according to Lineweaver-Burk to calculate the corresponding Km values. All error bars represent sd (n = 3).