An active role for tRNA in decoding beyond codon:anticodon pairing

Abstract

During transfer RNA (tRNA) selection, a cognate codon:anticodon interaction triggers a series of events that ultimately results in the acceptance of that tRNA into the ribosome for peptide-bond formation. High-fidelity discrimination between the cognate tRNA and near- and noncognate ones depends both on their differential dissociation rates from the ribosome and on specific acceleration of forward rate constants by cognate species. Here we show that a mutant tRNA(Trp) carrying a single substitution in its D-arm achieves elevated levels of miscoding by accelerating these forward rate constants independent of codon:anticodon pairing in the decoding center. These data provide evidence for a direct role for tRNA in signaling its own acceptance during decoding and support its fundamental role during the evolution of protein synthesis.

Fig. 1

Kinetic scheme for tRNA selection on the ribosome identifying the two stages of initial selection and proofreading. The scheme includes the relevant kinetically resolved steps (). EF-Tu is shown in different conformations in the GTP- and GDP-bound form.

Fig. 2

G24A tRNA^Trp variant accelerates forward rates of (A) GTPase activation and (B) accommodation on near-cognate codons. The top panels show representative time courses (at 2 μM ribosomes) of GTP hydrolysis and dipeptide formation for wild-type (solid lines, open symbols) and G24A (dashed lines, solid symbols) tRNA^Trp on cognate (circles), UGA (squares), or CGG (diamonds) programmed ribosomes. The middle panels show ribosome titrations and fits for rate-constant calculations. The bottom panels show calculated rate constants for wild-type (black) and G24A (white) tRNA^Trp. Each bar represents the average of two to four ribosome titration experiments and the error bars represent their standard deviations.

Fig. 3

G24A tRNA^Trp variant does not increase miscoding levels by slowing rejection from the ribosome. (A) Rate constants of rejection during proofreading (k₇) were calculated from the rate and extent of dipeptide formation for wild-type (wt) (black) and G24A (white) tRNA^Trp. Errors were calculated by standard error propagation. (B) Equilibrium dissociation constants (K_d’s) were measured by filter-binding for wild-type (solid lines, open symbols) and G24A tRNA (dashed lines, solid symbols) on cognate (circles), UGA (squares), or CGG (diamonds) programmed ribosomes.