Distinct functional classes of ram mutations in 16S rRNA

Abstract

During decoding, the ribosome selects the correct (cognate) aminoacyl-tRNA (aa-tRNA) from a large pool of incorrect aa-tRNAs through a two-stage mechanism. In the initial selection stage, aa-tRNA is delivered to the ribosome as part of a ternary complex with elongation factor EF-Tu and GTP. Interactions between codon and anticodon lead to activation of the GTPase domain of EF-Tu and GTP hydrolysis. Then, in the proofreading stage, aa-tRNA is released from EF-Tu and either moves fully into the A/A site (a step termed "accommodation") or dissociates from the ribosome. Cognate codon-anticodon pairing not only stabilizes aa-tRNA at both stages of decoding but also stimulates GTP hydrolysis and accommodation, allowing the process to be both accurate and fast. In previous work, we isolated a number of ribosomal ambiguity (ram) mutations in 16S rRNA, implicating particular regions of the ribosome in the mechanism of decoding. Here, we analyze a representative subset of these mutations with respect to initial selection, proofreading, RF2-dependent termination, and overall miscoding in various contexts. We find that mutations that disrupt inter-subunit bridge B8 increase miscoding in a general way, causing defects in both initial selection and proofreading. Mutations in or near the A site behave differently, increasing miscoding in a codon-anticodon-dependent manner. These latter mutations may create spurious favorable interactions in the A site for certain near-cognate aa-tRNAs, providing an explanation for their context-dependent phenotypes in the cell.

Keywords: EF-Tu; decoding; ribosome; tRNA; translation.

FIGURE 1.

Locations of ram mutations in 16S rRNA. Tertiary structure of the 16S rRNA, viewed from the subunit interface (A) and solvent (B) perspectives, and in the context of the 70S ribosome with bound ternary complex (C). Red spheres indicate positions of ram mutations. Small subunit proteins other than S4, S5, and S12 have been computationally removed for clarity. (SHDR) Shoulder domain, (PF) platform domain. (D) Zoomed-in view of intersubunit bridge B8. Hydrogen bonds that are lost upon bridge disruption are shown as dashed lines. (E) Zoomed-in view of the 30S A site showing the interactions of various 16S rRNA residues with A-tRNA and mRNA. For clarity, h18 and S12 are omitted from the foreground of this view. (Gray) 16S rRNA, (tan) 23S/5S rRNA, (magenta) unlabeled 50S proteins, other features as indicated. Figure based on PDB files 2WRN, 2WRO, 2WDG, and 2WDI.

FIGURE 2.

Effects of 16S A-site mutations on EF-Tu-dependent GTP hydrolysis and RF2-dependent termination. (A,B) 70S initiation complexes (70SIC) programmed with either cognate UUU or near-cognate CUU in the A site were rapidly mixed with EF-Tu•[γ³²P]GTP•Phe-tRNA^Phe, and rates of GTP hydrolysis were determined. Shown are apparent rates for cognate (A) and near-cognate (B) reactions plotted vs. 70SIC concentration. Data were fit to the equation k_app = k_cat[70SIC]/(K_M+[70SIC]), yielding the kinetic parameters shown in Table 1. (C) Ribosomes (0.2 µM) containing Ac[³⁵S]Met-tRNA^Met in the P site and codon UAA in the A site were rapidly mixed with RF2, and rates of Ac[³⁵S]Met-tRNA^Met hydrolysis were determined. Shown are apparent rates plotted as a function of RF2 concentration. Data were fit to the quadratic equation k_app = k_cat{(A + B + K_M) – [(A + B + K_M)² – 4AB]^1/2}/(2A), where A and B represent the total concentrations of ribosomal complex and RF2, respectively, in the reaction. This yielded the k_cat and K_M parameters shown in Table 2. Control, ○ and solid lines; C1054U, □ and long-dashed lines; C1054A, ⋄ and medium-dashed lines; C1200U, △ and short-dashed lines; G1491A, ▽ and dotted lines.

FIGURE 3.

Effects of 16S ram mutations on overall miscoding in various contexts. Control and mutant 70SICs (color-coded, as indicated) programmed with various near-cognate codons in the A site were mixed with the TC under conditions that facilitate reassembly of the TC, and apparent rates of miscoding were measured. Misincorporation rates for Phe (A, E, I), Tyr (B, F, J), Lys (C, G, K), and Glu (D, H, L) in the presence of various first-position (A–D) or third-position (E–L) codon-anticodon mismatches were compared. The codon programmed in the A site of each ribosome complex is indicated above each graph, with the mismatched base colored red. Anticodon sequences for each tRNA are indicated at the top of the figure. Data in panels A–J represent the mean ± SEM from three or more independent experiments. For panels K and L, rates were measured using a quench-flow machine, and reported values and their standard errors come from the curve fits.