Efficiency of GTP hydrolysis increases with increasing Mg

^{2+} concentration. (

*A* and

*B*) Time evolution of the [

^{3}H]GDP level after mixing of

with ribosomes programmed with noncognate

GAA (green squares) or cognate AAA (black circles) codon with no (

*A*) or 6 mM (

*B*) extra Mg

^{2+} addition (see also Fig. S1 for a complete set of mismatch experiments at 2 mM extra Mg

^{2+}). The initial [

^{3}H]GDP increase is determined by the rate of hydrolysis of [

^{3}H]GTP in ternary complex. The decrease is due to spontaneous exchange of [

^{3}H]GDP for unlabeled GTP on EF-Tu after GTP hydrolysis, and rapid regeneration of [

^{3}H]GTP. The slow initial increase of [

^{3}H]GDP in noncognate cases (green squares), is faster in high (

*B*) than in low (

*A*) Mg

^{2+} concentration, and much slower than in the cognate cases (black line). The noncognate and cognate curves in

*A* and

*B* were obtained in parallel experiments and jointly fitted for precise estimates of the noncognate

*k*_{cat}/

*K*_{m} values for GTP hydrolysis. (

*Insets*) [

^{3}H]GDP level for the cognate reaction as measured in quench-flow to obtain the cognate

*k*_{cat}/

*K*_{m} values for GTP hydrolysis. (

*C*) Efficiency of GTP hydrolysis,

*k*_{cat}/

*K*_{m}, for noncognate reading of

GAA codon (green squares) and cognate reading of AAA codon (black circles) at varying Mg

^{2+} concentration. Data represent weighted averages from at least two experiments ± propagated standard deviation.

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