Control of translation initiation involves a factor-induced rearrangement of helix 44 of 16S ribosomal RNA

Abstract

Initiation of translation involves recognition of the start codon by the initiator tRNA in the 30S subunit. To investigate the role of ribosomal RNA (rRNA) in this process, we isolated a number of 16S rRNA mutations that increase translation from the non-canonical start codon AUC. These mutations cluster to distinct regions that overlap remarkably well with previously identified class III protection sites and implicate both IF1 and IF3 in start codon selection. Two mutations map to the 790 loop and presumably act by inhibiting IF3 binding. Another cluster of mutations surrounds the conserved A1413(o)G1487 base pair of helix 44 in a region known to be distorted by IF1 and IF3. Site-directed mutagenesis in this region confirmed that this factor-induced rearrangement of helix 44 helps regulate initiation fidelity. A third cluster of mutations maps to the neck of the 30S subunit, suggesting that the dynamics of the head domain influences translation initiation. In addition to identifying mutations that decrease fidelity, we found that many P-site mutations increase the stringency of start codon selection. These data provide evidence that the interaction between the initiator tRNA and the 30S P site is tuned to balance efficiency and accuracy during initiation.

Fig. 1

Nucleotides identified in this study as important for initiation fidelity cluster to h44, the 790 loop and the neck region. A. Mutation sites overlap with class III protection sites. Positions of mutations identified in this study (red triangles); class III protection sites attributed to ligand-induced conformation changes (black squares; Moazed and Noller, 1987). B. Positions of mutations in h44 and in the 790 loop (image based on Selmer et al., 2006; PDB 2J02). C. Binding of IF1 to the 30S subunit distorts h44 where our mutations map. Blue ribbon, with IF1; grey ribbon, without IF1 (image based on Carter et al., 2001; PDB 1HR0 and 1J5E). D. Positions of mutations in the neck region (image based on Selmer et al., 2006; PDB 2J02).

Fig. 2

Effects of 16S rRNA mutations on the relative level of translation from AUC and ACG. Values correspond to the level of β-galactosidase expressed by specialized ribosomes from either SD*-AUC-lacZ (A) or SD*-ACG-lacZ (B) relative to SD*-AUG-lacZ. Grey bars, wild-type infC+ background; white bars, infC362 background. Values represent the quotient of two means ± standard error.

Fig. 3

Effect of mutations in the 790 loop on the binding of IF3-AF to the 30S subunit. Fluorescently labelled IF3 (IF3-AF; 1 μM) was incubated with each preparation of 30S subunits (1 μM) at 37°C for 20 min and then subjected to sucrose gradient sedimentation analysis. Fractions (0.5 ml) were collected and the fluorescence intensity (excitation 495 nm; emission 520 nm) of each was quantified to determine the distribution of IF3-AF in the gradient. The position of 30S subunits in the gradient, as identified by A260, is indicated.

Fig. 4

Effects of 16S rRNA mutations on the binding of IF1 or tRNA^fMet to the 30S subunit. A. Examples of experiments used to estimate the affinity of IF1 for mutant 30S subunits. [³⁵S]-IF1 (0.25 μM) was incubated with 30S subunits (various concentration) in buffer C [10 mM Tris-HCl (pH 7.5), 100 mM NH4Cl, 5 mM Mg(OAc)2, 6 mM β-ME and 0.03% Nikkol] at room temperature for 20 min. Microfiltration devices were used to separate free and bound [³⁵S]-IF1. The fraction of [³⁵S]-IF1 bound, averaged from at least three independent experiments, was plotted as a function of the concentration of 30S subunits, and the data were fit using KaleidaGraph. B. Examples of experiments to estimate the affinity of tRNA^fMet for mutant 30S subunits. 3′-[³²P] -tRNA^fMet (5 nM) was incubated with 30S subunits (various concentration) with a model mRNA containing an AUG start codon (m701; 3 μM) in buffer D [50 mM Tris-HCl (pH 7.5), 100 mM NH4Cl, 20 mM MgCl2, 6 mM β-ME] for 20 min at 37°C. The free and bound 3′-[³²P]-tRNA^fMet were separated by filtration through a bilayer of nitrocellulose and nylon membranes. The fraction of 3′-[³²P]-tRNA^fMet bound was plotted as a function of 30S subunit concentration, and the data were fit using KaleidaGraph. Equilibrium binding constants (KD) listed in Table 2 represent the mean ± SEM from three independent experiments.

Fig. 5

Effects of alternative base pairs at 1410–1490, 1413–1487 and 1414–1486 on the relative level of translation from AUC and ACG. Values correspond to the level of β-galactosidase expressed by specialized ribosomes from either SD*-AUC-lacZ (grey bars) or SD*-ACG-lacZ (white bars) relative to SD*-AUG-lacZ. Values represent the quotient of two means ± standard error.