title: On the pH dependence of class-1 RF-dependent termination of mRNA translation.
abstract: We have studied the pH dependence of the rate of termination of bacterial protein synthesis catalyzed by a class-1 release factor (RF1 or RF2). We used a classical quench-flow technique and a newly developed stopped-flow technique that relies on the use of fluorescently labeled peptides. We found the termination rate to increase with increasing pH and, eventually, to saturate at about 70 s(-1) with an apparent pKa value of about 7.6. From our data, we suggest that class-1 RF termination is rate limited by the chemistry of ester bond hydrolysis at low pH and by a stop-codon-dependent and pH-independent conformational change of RFs at high pH. We propose that RF-dependent termination depends on the participation of a hydroxide ion rather than a water molecule in the hydrolysis of the ester bond between the P-site tRNA and its peptide chain. We provide a simple explanation for why the rate of termination saturated at high pH in our experiments but not in those of others.
authors.affiliation: Department of Cell and Molecular Biology, Uppsala University, Biomedicinskt Centrum, Box 596, 75124 Uppsala, Sweden.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Indrisiunaite
authors.affiliation: Department of Cell and Molecular Biology, Uppsala University, Biomedicinskt Centrum, Box 596, 75124 Uppsala, Sweden.
authors.forename: Michael Y
authors.initials: MY
authors.lastname: Pavlov
authors.affiliation: Centre National de la Recherche Scientifique, FRE3630, University Paris Diderot Sorbonne Paris Cité Institut de Biologie Physico-Chimique, 13 Rue Pierre et Marie Curie, 75005 Paris, France.
authors.forename: Valérie
authors.initials: V
authors.lastname: Heurgué-Hamard
authors.affiliation: Department of Cell and Molecular Biology, Uppsala University, Biomedicinskt Centrum, Box 596, 75124 Uppsala, Sweden. Electronic address: ehrenberg@xray.bmc.uu.se.
authors.forename: Måns
authors.initials: M
authors.lastname: Ehrenberg
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D018388:Codon, Terminator / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D010446:Peptide Fragments / Q000378:metabolism; D010454:Peptide Termination Factors / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D018388:Codon, Terminator; D010446:Peptide Fragments; D010454:Peptide Termination Factors; D012333:RNA, Messenger; D012346:RNA, Transfer, Amino Acyl; D012343:RNA, Transfer
keywords: conformational change; fast kinetics; nucleophile; release factor; ribosome