The codon specificity of eubacterial release factors is determined by the sequence and size of the recognition loop

Abstract

The two codon-specific eubacterial release factors (RF1: UAA/UAG and RF2: UAA/UGA) have specific tripeptide motifs (PXT/SPF) within an exposed recognition loop shown in recent structures to interact with stop codons during protein synthesis termination. The motifs have been inferred to be critical for codon specificity, but this study shows that they are insufficient to determine specificity alone. Swapping the motifs or the entire loop between factors resulted in a loss of codon recognition rather than a switch of codon specificity. From a study of chimeric eubacterial RF1/RF2 recognition loops and an atypical shorter variant in Caenorhabditis elegans mitochondrial RF1 that lacks the classical tripeptide motif PXT, key determinants throughout the whole loop have been defined. It reveals that more than one configuration of the recognition loop based on specific sequence and size can achieve the same desired codon specificity. This study has provided unexpected insight into why a combination of the two factors is necessary in eubacteria to exclude recognition of UGG as stop.

FIGURE 1.

RF recognition loop codon specificity determinants. (A) The recognition loop regions of E. coli RF1 and RF2. The 10 loop residues are shown with the β-strands surrounding the recognition loop highlighted in black. The tripeptide motifs (PXT/SPF) are highlighted in gray. Specificity-determining positions are numbered, and the black line indicates the amino acids analyzed. (B) The RF1-RF2 recognition loop variants. The residues substituted are shown in black. (C) The codon-dependent peptidyl-tRNA hydrolysis activities of the RF1-RF2 recognition loop variants at the standard stop codons UAG, UAA, and UGA, and the sense codon UGG. Release activity is given in Δcpm of [³H]fMet released. The RF variants were assayed in triplicate with each experiment repeated twice to ensure consistent results between assays. RF2 is included as a positive control for UGA activity. The error bars represent the standard deviation of the mean. (D) The effect of overexpression of the RF1-RF2 recognition loop variants on termination efficiency at the Ra UGA Ra termination context in the E. coli suppressor strain CDJ64. (Top) A SDS polyacrylamide gel showing readthrough and termination products that reflect termination efficiencies. (Bottom) The calculated termination efficiencies derived from this gel experiment. (M) Marker; (RF endo) endogenous RFs.

FIGURE 2.

RF1 recognition loop residue substitutions affect codon recognition and specificity. (A) The RF1 SPFD recognition loop variants. Substitutions are shown in black. (B) The codon-dependent peptidyl-tRNA hydrolysis activities of the SPFD recognition loop variants at the standard stop codons UAG, UAA, and UGA, and the sense codon UGG. The RF variants were assayed and the data analyzed as described for Figure 1C. (C) Codon specificity of the RF1 SPFD(ItoR) recognition loop. The SPFD(ItoR) variant was assayed with the codons indicated and the data analyzed as for Figure 1C. Statistical significances, (**) P < 0.01, (***) P < 0.001 (Student's two-tailed t-test), were determined by comparison to the activity of RF1-SPFD(ItoR) with no codon.

FIGURE 3.

Codon recognition and specificity of the C. elegans MRF1 novel recognition loop. (A) A sequence alignment of the recognition loop regions of a selection of nonvertebrate MRF1 proteins. Identical amino acids are white on a black background, those similar are black on a gray background, and different amino acids are black on a white background. Full listings for each species are in Supplemental Table 1. (B, top) The recognition loop regions of E. coli RF1 and C. elegans MRF1. The β-strands surrounding the recognition loop are shown in black. The RF1-specific tripeptide (PXT) motif of E. coli RF1 is highlighted in gray. Specificity-determining positions are numbered. (Bottom) The RF1 C. elegans MRF1 recognition loop variants. Amino acid substitutions are shown in black, and deletions are boxed. (C) The codon-dependent peptidyl-tRNA hydrolysis activities of the RF1 C. elegans MRF1 recognition loop variants at the standard stop codons UAG, UAA, and UGA. The release activities are expressed as percentages of the release activity of RF1 for UAG and UAA, and RF2 for UGA. The RF variants were assayed and data analyzed as for Figure 1C. (D) The effect of overexpression of the RF1 C. elegans MRF1 anticodon loop variants on termination efficiency at the Ra UAG Ra termination context in the E. coli suppressor strain XA102. The RF variants were assayed in triplicate in three separate experiments to ensure consistent results between assays. The error bars represent the standard deviation of the mean. Statistical significances, (***) P < 0.001 (Student's two-tailed t-test), were determined by comparison to the activity of either RF1 or RF1ΔQG.

FIGURE 4.

The activity of PVND motif variants in the RF1 recognition loop. (Top) The RF1 PVND variants with shortened loop size as for C. elegans (A) or as for a typical RF (B). Amino acid substitutions are shown in black. (Middle) The codon-dependent peptidyl-tRNA hydrolysis activities of the RF1 PVND motif variants at the standard stop codons UAG, UAA, and UGA. The release activities are expressed as percentages of the release activity of RF1 for UAG and UAA, and RF2 for UGA. The RF variants were assayed and data analyzed as for Figure 1C. (Bottom) The effect of overexpression of the RF1 PVND variants on termination efficiency at the Ra UAG Ra termination context in the E. coli suppressor strain XA102. Replicates and analyses were as for Figure 3D.

FIGURE 5.

An explanation for how the recognition loops of RF1 and RF2 maintain specificity. (A) The recognition loop residues responsible for E. coli RF codon specificity. Residues that are important for determining the different specificities of RF1 and RF2 at the second and third positions of the stop codon are indicated. (B) The isolated and ribosome-bound structures of the RF1 and RF2 recognition loops RF1 (Graille et al. 2005; Laurberg et al. 2008) and RF2 (Vestergaard et al. 2001; Weixlbaumer et al. 2008) with key elements highlighted (residues) or annotated (helices and strands).