Steric complementarity in the decoding center is important for tRNA selection by the ribosome

Abstract

Accurate tRNA selection by the ribosome is essential for the synthesis of functional proteins. Previous structural studies indicated that the ribosome distinguishes between cognate and near-cognate tRNAs by monitoring the geometry of the codon-anticodon helix in the decoding center using the universally conserved 16S ribosomal RNA bases G530, A1492 and A1493. These bases form hydrogen bonds with the 2'-hydroxyl groups of the codon-anticodon helix, which are expected to be disrupted with a near-cognate codon-anticodon helix. However, a recent structural study showed that G530, A1492 and A1493 form hydrogen bonds in a manner identical with that of both cognate and near-cognate codon-anticodon helices. To understand how the ribosome discriminates between cognate and near-cognate tRNAs, we made 2'-deoxynucleotide and 2'-fluoro substituted mRNAs, which disrupt the hydrogen bonds between the A site codon and G530, A1492 and A1493. Our results show that multiple 2'-deoxynucleotide substitutions in the mRNA substantially inhibit tRNA selection, whereas multiple 2'-fluoro substitutions in the mRNA have only modest effects on tRNA selection. Furthermore, the miscoding antibiotics paromomycin and streptomycin rescue the defects in tRNA selection with the multiple 2'-deoxynucleotide substituted mRNA. These results suggest that steric complementarity in the decoding center is more important than the hydrogen bonds between the A site codon and G530, A1492 and A1493 for tRNA selection.

Keywords: EF-Tu; GTP hydrolysis; elongation factor Tu; kinetics; peptide bond; protein synthesis; rRNA; ribosomal RNA.

Figure 1. Interaction of the A site codon with the ribosome and equilibrium binding of EF-Tu ternary complex to the ribosome

(a) Interaction of the mRNA at positions +4 to +6 with the decoding center (PDB ID: 1IBM). Shown are mRNA bases (U1, U2 and U3), tRNA bases (A36, A35 and G34), 16S rRNA bases (A1493, A1492, G530 and C518) and residues from ribosomal protein S12 (Ser50 and Pro48). Hydrogen bonds are represented by the dotted lines. (b to d) Representative equilibrium binding curves for EF-Tu (H84A)•Phe-tRNA^Phe•GTP ternary complex binding to the ribosomal A site. Data are shown for the control mRNA (λ) and mRNAs with a single 2’-deoxynucleotide substitution at positions +4 (ν), +5 (σ), and +6 (υ). The data were fit to a one-site binding hyperbolic equation to determine K_D. (c) Representative binding curves for the control mRNA (λ), mRNAs with two 2’-deoxynucleotide substitutions at positions +4 and +5 (ν) or +5 and +6 (σ) and an mRNA with three 2’-deoxynucleotide substitutions at positions +4, +5 and +6 (υ). (d) Representative binding curves for the control mRNA (λ), an mRNA with a single 2’-fluoro substitution at position +5 (ν), an mRNA with two 2’-fluoro substitutions at positions +5 and +6 (σ), and an mRNA with three 2’-fluoro substitutions at positions +4, +5 and +6 (υ).

Figure 2. Rate of GTP hydrolysis by EF-Tu ternary complex

(a) Representative time course for ribosome-dependent GTP hydrolysis by EF-Tu ternary complex. Data are shown for the control mRNA (λ) and mRNAs with a single 2’-deoxynucleotide substitution at positions +4 (ν), +5 (σ), and +6 (υ). The data were fit to a single exponential equation to obtain the rate of GTP hydrolysis. (b) Representative GTP hydrolysis data for the control mRNA (λ), mRNAs with two 2’-deoxynucleotide substitutions at positions +4 and +5 (ν) or +5 and +6 (σ) and an mRNA with three 2’-deoxynucleotide substitutions at positions +4, +5 and +6 (υ). (c) Representative GTP hydrolysis data for the control mRNA (λ), an mRNA with a single 2’-fluoro substitution at position +5 (ν), an mRNA with two 2’-fluoro substitutions at positions +5 and +6 (σ), and an mRNA with three 2’-fluoro substitutions at positions +4, +5 and +6 (υ). (d) Graph showing the rate of GTP hydrolysis at varying concentration of ribosomes for the control mRNA (λ), an mRNA with two 2’-deoxynucleotide substitutions at positions +5 and +6 (σ), an mRNA with three 2’-deoxynucleotide substitutions at positions +4, +5 and +6 (ν), and an mRNA with three 2’-fluoro substitutions at positions +4, +5 and +6 (υ).

Figure 3. Rate of peptide bond formation

(a) Representative time course showing the kinetics of dipeptide formation. Ribosomes contained f[³⁵S]Met-tRNA^fMet in the P site and were reacted with EF-Tu•Phe-tRNA^Phe•GTP ternary complex. Data are shown for the control mRNA (λ) and mRNAs with a single 2’-deoxynucleotide substitution at positions +4 (ν), +5 (σ), and +6 (λ). The data were fit to a single exponential equation to obtain the rate of peptide bond formation. (b) Representative peptide bond formation data for the control mRNA (λ), mRNAs with two 2’-deoxynucleotide substitutions at positions +4 and +5 (ν) or +5 and +6 (σ) and an mRNA with three 2’-deoxynucleotide substitutions at positions +4, +5 and +6 (υ). (c) Representative peptide bond formation data for the control mRNA (λ), an mRNA with a single 2’-fluoro substitution at position +5 (ν), an mRNA with two 2’-fluoro substitutions at positions +5 and +6 (σ), and an mRNA with three 2’-fluoro substitutions at positions +4, +5 and +6 (υ). (d) Bar graph showing the rate of peptide bond formation with the 2′-deoxynucleotide and the 2′-fluoro substituted mRNAs. Error bars represent s.d. from two experiments.

Figure 4. Effect of paromomycin and streptomycin on the rates of GTP hydrolysis and peptide bond formation

(a) Bar graph showing the rates of GTP hydrolysis by EF-Tu ternary complex with the control mRNA and with the mRNA having three 2’-deoxynucleotide substitutions at positions +4, +5 and +6. Experiments were done in the absence of antibiotics (white bar), in the presence of paromomycin (grey bar) or in the presence of streptomycin (black bar). (b) Bar graph showing the rates of peptide bond formation with the control mRNA, with the mRNA having three 2’-deoxynucleotide substitutions at positions +4, +5 and +6, and with the mRNA having three 2’-fluoro substitutions at positions +4, +5 and +6. Labels for the bar graph are as indicated above. The asterisks are used to indicate that in the absence of antibiotics the rate of peptide bond formation was very slow with the triple 2’-deoxynucleotide substituted mRNA. (c) Bar graph showing the error frequency with 2’ -deoxynucleotide and 2’-fluoro substituted mRNAs. The asterisks are used to indicate that only negligible amounts of dipeptides were formed with the triple 2’-deoxynucleotide substituted mRNA. In all cases, the error bars represent s.d. from at least two experiments.