Real-time assay for testing components of protein synthesis

• Published in: Nucleic acids research Vol. 40; no. 12; p. e88
• Main Authors: Rosenblum, Gabriel, Chen, Chunlai, Kaur, Jaskiran, Cui, Xiaonan, Goldman, Yale E, Cooperman, Barry S
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01-07-2012
• Subjects: Cell-Free System; Fluorometry - methods; Green Fluorescent Proteins - biosynthesis; Luminescent Agents - analysis; Methods Online; Phenylalanine - metabolism; Protein Biosynthesis; Ribosomes - metabolism; RNA, Transfer, Amino Acyl - metabolism; RNA, Transfer, Phe - metabolism; Transcription, Genetic
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gks232

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.