Accuracy of genetic code translation and its orthogonal corruption by aminoglycosides and Mg2+ ions

Abstract

We studied the effects of aminoglycosides and changing Mg2+ ion concentration on the accuracy of initial codon selection by aminoacyl-tRNA in ternary complex with elongation factor Tu and GTP (T3) on mRNA programmed ribosomes. Aminoglycosides decrease the accuracy by changing the equilibrium constants of 'monitoring bases' A1492, A1493 and G530 in 16S rRNA in favor of their 'activated' state by large, aminoglycoside-specific factors, which are the same for cognate and near-cognate codons. Increasing Mg2+ concentration decreases the accuracy by slowing dissociation of T3 from its initial codon- and aminoglycoside-independent binding state on the ribosome. The distinct accuracy-corrupting mechanisms for aminoglycosides and Mg2+ ions prompted us to re-interpret previous biochemical experiments and functional implications of existing high resolution ribosome structures. We estimate the upper thermodynamic limit to the accuracy, the 'intrinsic selectivity' of the ribosome. We conclude that aminoglycosides do not alter the intrinsic selectivity but reduce the fraction of it that is expressed as the accuracy of initial selection. We suggest that induced fit increases the accuracy and speed of codon reading at unaltered intrinsic selectivity of the ribosome.

Figure 1.

Effects of paromomycin on the kinetics of GTP hydrolysis on ternary complexes reading cognate (UUC) and near-cognate (CUC) codons. Time courses of [³H]GDP accumulation after mixing ternary complexes [³H]GTP·EF-Tu·Phe-tRNA^Phe with excess of mRNA programmed 70S ribosomes with cognate codon UUC (black squares) or near-cognate codon CUC (red circles) in the A-site. Cognate and near-cognate reactions were conducted in parallel at 1.3 mM free Mg²⁺ concentration (high accuracy condition). [³H]GDP accumulation in fast time scale is shown in inserts. Actual rates, k, of GTP hydrolysis for T₃ reading UUC and CUC codons were obtained from fits of the corresponding curves (see M&M for details). Panel A: (no drug) k = 34 s⁻¹ for UUC and k = 0.024 s⁻¹ for CUC reading. Panel B: (with Paromomycin) k = 32 s⁻¹ for UUC and k = 1.3 s⁻¹ for CUC reading.

Figure 2.

Effects of [Mg²⁺] and paromomycin on the kinetic efficiency (k_cat/K_m) of cognate and near-cognate GTPase reactions. Log₁₀-values of k_cat/K_m of GTP hydrolysis in T₃ ([³H]GTP·EF-Tu·Phe-tRNA^Phe) reading cognate (UUC) or near-cognate (CUC) codonplotted as functions of [Mg²⁺] in the presence (, UUC; , CUC) or absence (, UUC; , CUC) of paromomycin.

Figure 3.

The effect of aminoglycosides on the efficiency-accuracy trade-off. Kinetic efficiency (k_cat/K_m)^c of the reaction of GTP hydrolysis on EF-Tu·GTP·Phe-tRNA^Phe ternary complex reading its cognate UUC codon was plotted versus accuracy of initial selection for the same ternary complex reading its near-cognate codons in the presence of paromomycin, gentamicin and neomycin or in their absence (inserts). The intercepts of straight lines with the accuracy axis (A-value when (k_cat/K_m)^c = 0) give the effective selectivity values compiled in Table 1. Panel A: CUC near-cognate codon; Panel B: UCC near-cognate codon; Panel C: UUA near-cognate codon.

Figure 4.

The effects of aminoglycosides on the dissociation-mean-time of GTPase-deficient cognate ternary complex. Time traces of dipeptide formation mirrow slow dissociation of GTPase deficient T₃ (EF-Tu(H84A)·GTP·Phe-tRNA^Phe), pre-bound to the A site of mRNA programmed ribosomes. The chase of GTPase deficient cognate T₃ by native cognate T₃ was conducted in the absence of aminoglycoside or in the presence of paromomycin, gentamicin or neomycin (see Materials and Methods for details).

Figure 5.

Four steps scheme of initial codon selection by ternary complex on mRNA programmed ribosomes. R₁ is the mRNA-programmed ribosome with empty A-site and peptidyl-tRNA in the P-site; T₃ is free ternary complex. Binding of T₃ to R₁ results in initial complex C₂ in which the anticodon of aa-tRNA in T₃ lacks contact with the mRNA codon. In complex C₃ aa-tRNA in T₃ is ‘bent’, its anticodon is in contact with the mRNA codon and monitoring bases A1492, A1493 and G530 are in ‘inactive conformation’. In complex C₄, the monitoring bases are activated and interact with the codon-anticodon helix promoting a ‘closure’ of the 30S subunit (see main text for details).