In Vitro Optimization Of Translational Activity With Modified Ribosomes

The cell-free protein synthesis (CFPS) kits were designed to perform protein synthesis with a minimal set of purified components, often derived from E. coli extracts. Hence, they offer the possibility to study translation steps as well as co-translational folding in vitro, at the single molecule level [1]. Such experiments require the ability to observe ribosomes tethered on a glass surface while they successfully produce a protein which has to remain bound to the ribosome. To this aim, a biotin anchor and fluorescent dyes have to be attached to the ribosomes and DNA templates were designed to inhibit the release step.First, to optimize the translational performances of our modified ribosomes, we measured their activity with ribosome depleted CFPS, by monitoring the in vitro production of the fast-maturating emerald GFP fluorescent reporter. Therefore, we showed that neither the addition of a biotin anchor nor the labeling procedure inhibits the ribosomal activity. In addition, our system requires the SecM arrest sequence to successfully inhibit the release of the nascent chain. Thus, in an attempt to optimize the non-release efficiency of our DNA templates, we quantified the modified ribosomes carrying a fluorescent protein with fluorescent correlation spectroscopy (FCS) and anisotropy measurements.Finally, the optimization of our ribosomes and DNA templates led us to fully understand and control the preparation steps of sample to further study co-translational folding at the single molecule level.1.Katranidis, A., et al., Fast biosynthesis of GFP molecules: a single-molecule fluorescence study. Angew Chem Int Ed Engl, 2009. 48(10): p. 1758-61.