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.
