On the pH Dependence of Class-1 RF-Dependent Termination of mRNA Translation
2015 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, no 9, 1848-1860 p.Article in journal (Refereed) Published
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 pK(a) 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.
Place, publisher, year, edition, pages
2015. Vol. 427, no 9, 1848-1860 p.
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:uu:diva-255073DOI: 10.1016/j.jmb.2015.01.007ISI: 000353929400006PubMedID: 25619162OAI: oai:DiVA.org:uu-255073DiVA: diva2:821760
FunderKnut and Alice Wallenberg FoundationSwedish Research Council