Class-1 translation termination factors: invariant GGQ minidomain is essential for release activity and ribosome binding but not for stop codon recognition

Previously, we have shown that all class-1 polypeptide release factors (RFs ) share a common glycine-glycine-glutamine (GGQ) motif, which is critical f or RF activity. Here, we subjected to site-directed mutagenesis two invaria nt amino acids, GIn185 and Arg189, situated in the GGQ minidomain of human eRF1, followed by determination of RF activity and the ribosome binding cap acity for mutant eRF1. We show that replacement of GIn185 with polar amino acid residues causes partial inactivation of RF activity; GIn185Ile, Arg189 Ala and Arg189Gln mutants are completely inactive; all mutants that retain partial RF activity respond similarly to thr stop codons. We suggest that l oss of RF activity for GIn185 and Arg189 mutants is caused by distortion of the conformation of the GGQ minidomain but not by damage of the stop codon recognition site of eRF1. Our data are inconsistent with the model postula ting direct involvement of GIn185 side chain in orientation of water molecu le toward peptidyl-tRNA ester bond at the ribosomal peptidyl transferase ce ntre. Most of the GIn185 mutants exhibit reduced ability to bind to the rib osome, probably, to rRNA and/or (peptidyl)-tRNA(s). The data suggest that t he GGQ motif is implicated both in promoting peptidyl-tRNA hydrolysis and b inding to the ribosome.