Specific Interaction between EF-G and RRF and Its Implication for GTP-Dependent Ribosome Splitting into Subunits

Abstract

After termination of protein synthesis, the bacterial ribosome is split into its 30S and 50S subunits by the action of ribosome recycling factor (RRF) and elongation factor G (EF-G) in a guanosine 5'-triphosphate (GTP)-hydrolysis-dependent manner. Based on a previous cryo-electron microscopy study of ribosomal complexes, we have proposed that the binding of EF-G to an RRF-containing posttermination ribosome triggers an interdomain rotation of RRF, which destabilizes two strong intersubunit bridges (B2a and B3) and, ultimately, separates the two subunits. Here, we present a 9-A (Fourier shell correlation cutoff of 0.5) cryo-electron microscopy map of a 50S x EF-G x guanosine 5'-[(betagamma)-imido]triphosphate x RRF complex and a quasi-atomic model derived from it, showing the interaction between EF-G and RRF on the 50S subunit in the presence of the noncleavable GTP analogue guanosine 5'-[(betagamma)-imido]triphosphate. The detailed information in this model and a comparative analysis of EF-G structures in various nucleotide- and ribosome-bound states show how rotation of the RRF head domain may be triggered by various domains of EF-G. For validation of our structural model, all known mutations in EF-G and RRF that relate to ribosome recycling have been taken into account. More importantly, our results indicate a substantial conformational change in the Switch I region of EF-G, suggesting that a conformational signal transduction mechanism, similar to that employed in transfer RNA translocation on the ribosome by EF-G, translates a large-scale movement of EF-G's domain IV, induced by GTP hydrolysis, into the domain rotation of RRF that eventually splits the ribosome into subunits.

Distinct functions of elongation factor G in ribosome recycling and translocation

“…The interaction of RRF with the ribosome has been studied by directed hydroxyl radical footprinting on ribosomal RNA (Lancaster et al 2002), by cryo-EM (Agrawal et al 2004;Gao et al 2005;Barat et al 2007;Gao et al 2007), and by X-ray crystallography (Wilson et al 2005;Borovinskaya et al 2007;Weixlbaumer et al 2007;Pai et al 2008). RRF is bound at the interface between the subunits where it interacts mainly with the 50S subunit.…”

“…Bridge 2a is most important for holding the subunits together, as the deletion of H69 destabilizes subunit interaction considerably (Ali et al 2006). A displacement of H69 by RRF bound to 70S ribosomes from Escherichia coli has been observed (Gao et al 2005;Wilson et al 2005;Borovinskaya et al 2007;Gao et al 2007), whereas on ribosomes from Thermus thermophilus the binding of RRF did not have this effect (Weixlbaumer et al 2007). These observations suggest that EF-G, driven by GTP hydrolysis and, as shown here, by Pi release induces a movement of RRF that, in turn, displaces H69 and possibly other bridges, thereby destabilizing the interactions between the subunits such that disassembly is promoted.…”

“…These observations suggest that EF-G, driven by GTP hydrolysis and, as shown here, by Pi release induces a movement of RRF that, in turn, displaces H69 and possibly other bridges, thereby destabilizing the interactions between the subunits such that disassembly is promoted. A movement of domain II of RRF, which is flexibly attached to domain I, seems to play an important role (Gao et al 2005(Gao et al , 2007.…”

“…There is evidence, however, indicating that the two factors interact on the ribosome. Interactions have been demonstrated by mutational analysis of EF-G and RRF (Ito et al 2002), by monitoring factor binding to the ribosome (Seo et al 2004), or by cryo-EM reconstructions of complexes of 50S ribosomal subunits with the two factors (Gao et al 2005(Gao et al , 2007. According to these data, the interaction between the two factors involves residues in domain III of EF-G and the hinge region in RRF as well as in domain IV of EF-G and domain II of RRF.…”

“…Pi release presumably induces a further conformational change of EF-G that is essential for ribosome disassembly to take place, in analogy to the initiation of the power stroke of motor ATPases by Pi release (Keller and Bustamante 2000). The conformational change appears to involve changes of the switch I and II regions in the G domain (Gao et al 2007) that change the conformation upon loss of interactions with the g-phosphate and relay the change to the neighboring domains. The low rate of ribosome disassembly, compared with unlocking in translocation (30 sec…”