Translation in real-time: Determining the activity of modified components of the protein synthesis machinery

Site-specific labeling of ribosomes and tRNAs allows mechanistic investigation of translation using single-molecule approaches. However, quality control of labeled components can be a laborious and cumbersome process. Here we develop a novel assay that allows straightforward determination of the activity of fluorescently labeled translational components. This assay follows translation in real-time, using cell-free expression of a GFP variant. The activity of fluorescently labeled ribosomes and tRNAs was tested in order to determine which fluorescent dye interferes least with translation activity.

HFSP Long-Term Fellow Gabriel Rosenblum and colleagues
authored on Tue, 17 April 2012

Protein synthesis is essential for all living organisms and is being investigated at many levels. Fluorescent labeling of translational components allows real-time monitoring of specific reactions that elucidate the structural dynamics and mechanistic aspects of the process. However, the use of fluorescent labeled components raises the question of whether introduction of exogenous labels affects the processes under study. Current assays of protein synthesis are often cumbersome, typically involving aliquot removal and subsequent point-by-point determinations. A novel radioactive-free assay of cell-free protein synthesis is described, where formation of the fluorescent protein Emerald GFP (EmGFP) is monitored in real-time. The continuous fluorescence signal is collected as the reaction is occurring using low sample volume (10 µl) in a high-throughput format, offering a simpler and more rapid approach over existing methods. Endogenous ribosomes and aminoacylated tRNAs were replaced by their exogenous, labeled counterparts, and the effect of labeling was assessed. While the activity of a labeled ribosome is not compromised, a more careful consideration is required to assess the effect of the label on the tRNA as it traverses the ribosome while dynamically forming and breaking contacts. In this case, the extent of activity loss correlates with a combination of steric bulk and hydrophobicity of the fluorophore.

Reference

Real-time assay for testing components of protein synthesis. Gabriel Rosenblum, Chunlai Chen, Jaskiran Kaur, Xiaonan Cui, Yale E. Goldman and Barry S. Cooperman.doi: 10.1093/nar/gks232.

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