Characterization of 16S rRNA mutations that decrease the fidelity of translation initiation

Abstract

In bacteria, initiation of translation is kinetically controlled by factors IF1, IF2, and IF3, which work in conjunction with the 30S subunit to ensure accurate selection of the initiator tRNA (fMet-tRNA(fMet)) and the start codon. Here, we show that mutations G1338A and A790G of 16S rRNA decrease initiation fidelity in vivo and do so in distinct ways. Mutation G1338A increases the affinity of tRNA(fMet) for the 30S subunit, suggesting that G1338 normally forms a suboptimal Type II interaction with fMet-tRNA(fMet). By stabilizing fMet-tRNA(fMet) in the preinitiation complex, G1338A may partially compensate for mismatches in the codon-anti-codon helix and thereby increase spurious initiation. Unlike G1338A, A790G decreases the affinity of IF3 for the 30S subunit. This may indirectly stabilize fMet-tRNA(fMet) in the preinitiation complex and/or promote premature docking of the 50S subunit, resulting in increased levels of spurious initiation.

FIGURE 1.

Mutation G1338A increases the affinity of tRNA^fMet for the 30S subunit P site. Wild-type (○) or mutant 30S subunits harboring G1338A (△) or A790G (□) were incubated at various concentrations with a model mRNA (containing an AUG codon; 2 μM) and 3′-[³²P]-tRNA^fMet. Then, the fraction of 3′-[³²P]-tRNA^fMet bound was determined by filtration through a bilayer of nitrocellulose and nylon membranes (see Materials and Methods). Equilibrium binding constants (K_D) listed in Table 2 come from at least three independent binding experiments like the one shown here.

FIGURE 2.

Mutation A790G decreases the affinity of IF3 for the 30S subunit. (A) Fluorescently labeled IF3 (IF3-AF) was incubated with mutant or wild-type 30S subunits, with 50S subunits, or in absence of subunits (as indicated), and the reactions were subjected to sucrose gradient sedimentation analysis. To determine the distribution of IF3-AF in each gradient, 0.5 mL fractions were collected and the fluorescence intensity (excitation 495 nm; emission 520 nm) of each fraction was quantified. To determine the distribution of ribosomal subunits in each gradient, A₂₆₀ was monitored during the fractionation process using an in-line UV detector. (B) Fluorescence anisotropy was used to estimate the affinity of IF3-AF for 30S subunits without (○) or with mutation G1338A (△) or A790G (□). Subunits at various concentrations were incubated with 10 nM IF3-AF, and fluorescence anisotropy was measured. Equilibrium binding constants (K_D) listed in Table 2 come from at least three independent binding experiments like those shown here.