Interaction of ribosome recycling factor and elongation factor EF-G with E-coli ribosomes studied by the surface plasmon resonance technique

Background: Ribosome recycling factor (RRF), in concert with elongation fac tor EF-G, is required for disassembly of the post-termination complex of a ribosome after the release of polypeptides. How RRF dissociates the complex has long been puzzling. Crystal structures of RRF molecules have been solv ed recently and shown to mimic a transfer RNA (tRNA) shape, which prompted us to examine whether RRF binds to the ribosome as tRNA does. Results: The formation of ribosome complexes on the surface-coupled RRF and elongation factor EF-G of Escherichia coli was monitored in real time with a BIACORE 2000 instrument based on the surface plasmon resonance technique . RRF interacted with 70S ribosomes as well as 50S and 30S subunits, althou gh it interacted preferentially with 50S subunits, which was clearly seen u nder high but physiological ionic conditions. This 50S interaction was dimi nished by a single amino acid substitutions for Arg132 of RRF, which did no t appreciably affect the protein folding but nullified the activity in vivo and in vitro. Moreover, a set of antibiotics that inhibited the RRF-50S in teraction were also inhibitory to the polysome breakdown activity of RRF in vitro. The BIACORE technique also worked very well in demonstrating the ac tion of the antibiotics thiostrepton and fusidic acid, which are inhibitory to the RRF function by freezing the pre- and post-translocation intermedia tes catalysed by EF-G. Conclusions: These results suggest that the preferential interplay of RRF w ith the 50S subunit may be of biological significance, probably reflecting the mode of RRF action. The BIACORE technique proved useful for real-time m onitoring of the interaction between the ribosome and translation factors, as well as for screening of potential inhibitors for ribosome recycling fac tor.