Functional elucidation of a key contact between tRNA and the large ribosomal subunit rRNA during decoding

Abstract

The selection of cognate tRNAs during translation is specified by a kinetic discrimination mechanism driven by distinct structural states of the ribosome. While the biochemical steps that drive the tRNA selection process have been carefully documented, it remains unclear how recognition of matched codon:anticodon helices in the small subunit facilitate global rearrangements in the ribosome complex that efficiently promote tRNA decoding. Here we use an in vitro selection approach to isolate tRNA(Trp) miscoding variants that exhibit a globally perturbed tRNA tertiary structure. Interestingly, the most substantial distortions are positioned in the elbow region of the tRNA that closely approaches helix 69 (H69) of the large ribosomal subunit. The importance of these specific interactions to tRNA selection is underscored by our kinetic analysis of both tRNA and rRNA variants that perturb the integrity of this interaction.

FIGURE 1.

In vitro selection scheme. The randomized tRNA pool (A) was constructed by mutating residues 10–32 and 38–67 at a 6% frequency (white residues). The anticodon loop and acceptor stem were left unrandomized (shaded residues). A representative round of selection (B) begins when the randomized tRNA pool is aminoacylated in the presence of EFTu and mixed with ribosome complexes. Ternary complexes able to catalyze the reaction on the UGA codon are isolated with paramagnetic streptavidin particles. The now enriched pool of tRNA variants is generated via RT-PCR and T7 transcription, and the cycle iterated again.

FIGURE 2.

Activity summary for nine rounds of in vitro selection (p0–p9). tRNA pools corresponding to the nine rounds of selection were evaluated (A) for the amount of dipeptide (fMet-Trp) formed on cognate (UGG)-programmed ribosome complexes and (B) for the amount of miscoding (calculated as a function of dipeptide formed on near–cognate (UGA) versus cognate (UGG)–ribosome complexes).

FIGURE 3.

Characterization of potent miscoding variants isolated by in vitro selection. The mutation frequency map (A) shows the frequency of mutation in fifty (n = 50) sequenced clones from pool 6. Nucleotides in black were not mutagenized in the experimental design. Colored nucleotides have a mutation frequency between 0% and 15% (blue), 15% and 25% (green), 25% and 49% (yellow), and >50% (red). (B) T7 transcribed tRNAs and the purified native (n) molecules were evaluated to determine the amount dipeptide formed on near-cognate (UGA)–programmed ribosome complexes after a 1-min incubation. The T7 transcripts (light gray) were assayed in HiFi buffer supplemented to 7 mM MgCl₂ and the native tRNAs (dark gray) in HiFi buffer.

FIGURE 4.

Structural analysis of variant tRNAs. (A) Ten percent PAGE in-line probing analysis where the indicated variant tRNAs were either incubated for 0 or 72 h at 25°C. Numbers highlight positions of interest in the cleavage pattern. Product identity was determined by comparison to a hydroxyl-mediated cleavage ladder (OH). (B) Quantitation of selected positions was performed using ImageQuant where the intensity of a band was normalized over the entire lane to account for loading differences and then normalized to cleavage at the same position (position 34 of the anticodon) for all variants tested. (C) Ten percent PAGE of T1 protection assay where the indicated variant tRNAs were either incubated with or without T1 nuclease. Numbers highlight positions of interest in the cleavage pattern. Product identity was determined by comparison to a hydroxyl-mediated cleavage ladder (OH). (D) Visual representation of structural data for the miscoding tRNA mutants. Sites of protection (blue circles) and enhanced cleavage (red circles) for the in-line probing assay and protection (green circles) for the T1 analysis are represented on a canonical tRNA clover-leaf structure (dots in the D and variable loops represent sites of varying length among tRNAs). Inclusion of sites was determined by comparison of individual positions to the WT molecule; the threshold for inclusion was a 2.5× difference from WT. Position 10 in all variants is protected in both assays.

FIGURE 5.

Contribution of large subunit rRNA components to tRNA selection. (A) The interactions between helix 69 (H69, teal) and a post-accomodation A-site tRNA on the ribosome are shown. The tip of H69 (residue A1913) is in close proximity to the D stem of the tRNA and the decoding center. tRNA residues highlighted (blue, green, or red) follow the same pattern as in Figure 4D. mRNA is represented in orange. E site (light pink) and P site (light brown) tRNAs are represented on the left for orientation. (B) Rates of accommodation (k₅) for the formation of fMet-Trp dipeptide for WT, G24A, G59A, and 24/27/59 tRNA^Trp with WT and A1913U ribosomes on UGG (black bars) and UGA (gray bars) codons.