title: A simple real-time assay for in vitro translation.
abstract: A high-throughput assay for real-time measurement of translation rates in cell-free protein synthesis (SNAP assay) is described. The SNAP assay enables quantitative, real-time measurement of overall translation rates in vitro via the synthesis of O(6)-alkylguanine DNA O(6)-alkyltransferase (SNAP). SNAP production is continuously detected by fluorescence produced by the reaction of SNAP with a range of quenched fluorogenic substrates. The capabilities of the assay are exemplified by measurements of the activities of Escherichia coli MRE600 ribosomes and fluorescently labeled E. coli mutant ribosomes in the PURExpress translation system and by determination of the 50% inhibitory concentrations (IC50) of three common macrolide antibiotics.
authors.affiliation: Department of Chemistry, Stanford University, Stanford, California 94305, USA.
authors.forename: Mark C
authors.initials: MC
authors.lastname: Capece
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305, USA.
authors.forename: Guy L
authors.initials: GL
authors.lastname: Kornberg
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305, USA.
authors.forename: Alexey
authors.initials: A
authors.lastname: Petrov
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305, USA puglisi@stanford.edu.
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Puglisi
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000096:biosynthesis; D004926:Escherichia coli; D006801:Humans; D018942:Macrolides / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics
chemical_list: D018942:Macrolides; D011500:Protein Synthesis Inhibitors; D019883:Alkyl and Aryl Transferases
keywords: IC50; SNAP; assay; in vitro translation; ribosome activity; single-molecule fluorescence; translation rate