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. 2013 Jun 25;9:678. doi: 10.1038/msb.2013.31

Figure 1.

Figure 1

Reconstituted ribosomes synthesize active firefly luciferase (Fluc). (A) Basic steps of conventional ribosome reconstitution methods are illustrated (Nierhaus, 1990). Ribosomal components (e.g., 23S rRNA, 5S rRNA, and TP50) are first prepared from the native subunits of tightly coupled 70S ribosomes. Then, ribosomal subunits are reconstituted in defined conditions from mature rRNA and proteins. Finally, in a separate reaction, the activity of assembled subunits (denoted with ‘R’) is assessed by directly adding the reconstitution reaction to our ETTA system. (B) The protein synthesis activities of 0.1 μM native 30S subunits or conventional 30S reconstitution reactions having 0.1 μM ribosomal components (R30S) with 0.2 μM native 50S subunits in an ETTA reaction. Batch reactions were carried out for 2 h at 30°C, where Fluc synthesis saturates. Reconstitutions in the absence of 16S rRNA or TP30 did not yield active particles. (C) The protein synthesis activities of 0.1 μM native 50S subunits or conventional 50S reconstitution reactions having 0.1 μM ribosomal components (R50S) with 0.2 μM native 30S subunits in an ETTA reaction. Batch reactions were carried out for 2 h at 30°C. Reconstitutions in the absence of 23S rRNA and 5S rRNA or TP50 did not yield active particles. Yield differences between the control reactions in B and C are not due to run-to-run variability, which is shown by the standard deviation bars and is low. Rather, the changes are caused by using different ratios of 30S to 50S subunits to ensure an excess of the non-assembled ribosomal subunit. (D) The activities of 50S reconstitution reactions having 0.1 μM ribosomal components (R50S) with 0.2 μM native 30S subunits were compared for different salt, temperature, and magnesium conditions. Angle brackets indicate two-step reactions. The final yield of Fluc after 2h batch incubations in an ETTA reaction is shown. Negative control reactions (e.g., TP50, 30S subunit) gave immeasurable results overall conditions (not shown). Values in all panels show means with error bars representing standard deviations (s.d.) of at least eight independent experiments.