Single-step in vitro ribosome reconstitution mediated by two GTPase factors, EngA and ObgE

When bacterial ribosomes are assembled in vitro, manipulation of incubation temperature and magnesium ion concentration have been an essential procedure, which is a crucial step for the assembly of active large subunits. The present study tackles with this issue to develop a single-step procedure, which can be performed in a physiological condition. We found that GTPase factors EngA and ObgE can complement the changes in temperature and magnesium ion concentrations. In the presence of these factors, ribosome assembly can proceed under physiological conditions, with magnesium ion concentrations below 10 mM, potassium concentrations around 100 mM, and temperatures of 37 °C. Both the ribosome assembly and translation processes were successfully integrated in the reconstituted cell-free protein synthesis system. Furthermore, we found that these GTPase factors can reassemble the ribosomes to an active state, whose structure was disrupted by EDTA chelation of magnesium ions, indicating that these two factors can reversibly induce the ribosome structure to an intact state. The findings are essential for the bottom-up construction of synthetic cells.