Studies of translational misreading in vivo show that the ribosome very efficiently discriminates against most potential errors

Abstract

Protein synthesis must rapidly and repeatedly discriminate between a single correct and many incorrect aminoacyl-tRNAs. We have attempted to measure the frequencies of all possible missense errors by tRNA , tRNA and tRNA . The most frequent errors involve three types of mismatched nucleotide pairs, U•U, U•C, or U•G, all of which can form a noncanonical base pair with geometry similar to that of the canonical U•A or C•G Watson-Crick pairs. Our system is sensitive enough to measure errors at other potential mismatches that occur at frequencies as low as 1 in 500,000 codons. The ribosome appears to discriminate this efficiently against any pair with non-Watson-Crick geometry. This extreme accuracy may be necessary to allow discrimination against the errors involving near Watson-Crick pairing.

Keywords: Escherichia coli; mistranslation; non-Watson–Crick base pairs; protein synthesis; β-galactosidase.

FIGURE 1.

Error frequencies by tRNA on mutants of codon 537 vary by over two orders of magnitude. The activity of mutants with the indicated codons in place of Glu codon 537 are shown relative to the activity of wild-type β-galactosidase (error bars, SEM). The insets use greatly expanded y-axis to represent the much lower activities of the 10 indicated mutants. (A) Comparison of mutant activities in the wild-type (XAc) and hyperaccurate (rpsL) genetic backgrounds. (B) Comparison of activities in wild-type and error-prone (rpsD) backgrounds.

FIGURE 2.

The activities of mutants of Asp codon 201 show that many codons exhibit high levels of functional replacement. The activities of the D201 mutants relative to wild type are shown as in Figure 1.

FIGURE 3.

The activities of mutants of Tyr codon 503 indicate significant second- and wobble-position errors. The activities of the Y503 mutants relative to wild type are shown as in Figure 1.

FIGURE 4.

The pattern of tRNA misreading involves two types of base mismatches. Each codon is represented by a small circle; filled circles connected by lines are all recognized by a single tRNA, with the anticodon wobble nucleotide of each tRNA identified immediately to the right using the standard convention (Limbach et al. 1994). The termination codons are represented by unfilled circles and lines connect those recognized by each of the peptide termination factors (PRF1 or PRF2). Misreading events in the wild-type background identified here or previously by Kramer and Farabaugh (2007) are shown as dashed arrows. The thickness of the arrow indicates relative frequency of the events (thick >10^–4 per codon; thin <<10^–4 per codon); for misreading of Glu codons GAA/GAG by tRNA, the frequency of misreading could not be more than 4 × 10^–4 per codon. The arrows are labeled to indicate the position of the mismatch and color coded for the nature of the mismatched base pair, as shown.