Mutations in conserved regions of ribosomal RNAs decrease the productive association of peptide-chain release factors with the ribosome during translation termination

Early studies provided evidence that peptide-chain release factors (RFs) bi nd to both ribosomal subunits and trigger translation termination. Although many ribosomal proteins have been implicated in termination, very few data present direct biochemical evidence for the involvement of rRNA. Particula rly absent is direct evidence for a role of a large subunit rRNA in RF bind ing. Previously we demonstrated in vitro that mutations in Escherichia coli rRNAs, known to cause nonsense codon readthrough in vivo, reduce the effic iency of RF2-driven catalysis of peptidyl-tRNA hydrolysis. This reduction w as consistent with the idea that in vivo defective termination at the mutan t ribosomes contributes to the readthrough. Nevertheless, other explanation s were also possible, because still missing was essential biochemical evide nce for that idea, namely, decrease in productive association of RFs with t he mutant ribosomes. Here we present such evidence using a new realistic in vitro termination assay. This study directly supports in vivo involvement in termination of conserved rRNA regions that also participate in other tra nslational events. Furthermore, this study provides the first strong eviden ce for involvement of large subunit rRNA in RF binding, indicating that the same rRNA region interacts with factors that determine both elongation and termination of translation. (C) 2000 Societe francaise de biochimie et bio logie moleculaire / Editions scientifiques et medicales Elsevier SAS.