title: Real-time assay for testing components of protein synthesis.
abstract: We present a flexible, real-time-coupled transcription-translation assay that involves the continuous monitoring of fluorescent Emerald GFP formation. Along with numerical simulation of a reaction kinetics model, the assay permits quantitative estimation of the effects on full-length protein synthesis of various additions, subtractions or substitutions to the protein synthesis machinery. Since the assay uses continuous fluorescence monitoring, it is much simpler and more rapid than other assays of protein synthesis and is compatible with high-throughput formats. Straightforward alterations of the assay permit determination of (i) the fraction of ribosomes in a cell-free protein synthesis kit that is active in full-length protein synthesis and (ii) the relative activities in supporting protein synthesis of modified (e.g. mutated, fluorescent-labeled) exogenous components (ribosomes, amino acid-specific tRNAs) that replace the corresponding endogenous components. Ribosomes containing fluorescent-labeled L11 and tRNAs labeled with fluorophores in the D-loop retain substantial activity. In the latter case, the extent of activity loss correlates with a combination of steric bulk and hydrophobicity of the fluorophore.
authors.affiliation: The Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Rosenblum
authors.forename: Chunlai
authors.initials: C
authors.lastname: Chen
authors.forename: Jaskiran
authors.initials: J
authors.lastname: Kaur
authors.forename: Xiaonan
authors.initials: X
authors.lastname: Cui
authors.forename: Yale E
authors.initials: YE
authors.lastname: Goldman
authors.forename: Barry S
authors.initials: BS
authors.lastname: Cooperman
mesh_terms: D002474:Cell-Free System; D005470:Fluorometry / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D049408:Luminescent Agents / Q000032:analysis; D010649:Phenylalanine / Q000378:metabolism; D014176:Protein Biosynthesis; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D049408:Luminescent Agents; D012346:RNA, Transfer, Amino Acyl; D012360:RNA, Transfer, Phe; D049452:Green Fluorescent Proteins; D010649:Phenylalanine