title: Two distinct components of release factor function uncovered by nucleophile partitioning analysis.
abstract: During translation termination, release factor (RF) 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 RF. Here we describe pre-steady-state kinetic and nucleophile competition experiments to examine RF contributions to the rate and specificity of peptide release. We find that while unacylated tRNA stimulates release in a nondiscriminating 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 RFs make two distinct contributions to catalysis--a relatively nonspecific activation of the catalytic center and specific selection of water as a nucleophile facilitated by Q235.
authors.affiliation: Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Shaw
authors.forename: Rachel
authors.initials: R
authors.lastname: Green
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D006868:Hydrolysis; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010443:Peptide Chain Termination, Translational / Q000502:physiology*; D010454:Peptide Termination Factors / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D017434:Protein Structure, Tertiary; D012270:Ribosomes / Q000378:metabolism; D016415:Sequence Alignment; D014867:Water / Q000737:chemistry / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; C489005:prfA protein, E coli; D014867:Water