Two distinct components of release factor function uncovered by nucleophile partitioning analysis

摘要

During translation termination, release factor protein catalyzes a hydrolytic reaction in the large subunit peptidyl transferase center to release the finished polypeptide chain. While the mechanism of catalysis of peptide release remains obscure, important contributing factors have been identified including conserved active site nucleotides and a GGQ tripeptide motif in the release factor. Here we describe pre-steady-state kinetic and nucleophile competition experiments to examine release factor contributions to the rate and specificity of peptide release. We find that while unacylated tRNA stimulates release in a non-discriminating manner, RF1 is very specific for water. Further analysis reveals that amino acid Q235 of the RF1 GGQ motif is critical for the observed specificity. These data lead to a model where release factors make two distinct contributions to catalysis – a relatively non-specific activation of the catalytic center and specific selection of water as a nucleophile facilitated by Q235.