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.
