New insights into the enzymatic role of EF-G in ribosome recycling

Abstract

During translation, elongation factor G (EF-G) plays a catalytic role in tRNA translocation and a facilitative role in ribosome recycling. By stabilizing the rotated ribosome and interacting with ribosome recycling factor (RRF), EF-G was hypothesized to induce the domain rotations of RRF, which subsequently performs the function of splitting the major intersubunit bridges and thus separates the ribosome into subunits for recycling. Here, with systematic mutagenesis, FRET analysis and cryo-EM single particle approach, we analyzed the interplay between EF-G/RRF and post termination complex (PoTC). Our data reveal that the two conserved loops (loop I and II) at the tip region of EF-G domain IV possess distinct roles in tRNA translocation and ribosome recycling. Specifically, loop II might be directly involved in disrupting the main intersubunit bridge B2a between helix 44 (h44 from the 30S subunit) and helix 69 (H69 from the 50S subunit) in PoTC. Therefore, our data suggest a new ribosome recycling mechanism which requires an active involvement of EF-G. In addition to supporting RRF, EF-G plays an enzymatic role in destabilizing B2a via its loop II.

A Possible Role of the Full-Length Nascent Protein in Post-Translational Ribosome Recycling

“…However RRF could not split pre-formed 70S-EFG-GTP complex and addition of unfolded protein to this after 7 min resulted in ribosome dissociation at as fast a rate as it does to free 70S (Fig 5B [D] ). These results go in favor of the previous reports where structural studies revealed that RRF, EFG share some overlapping binding sites on the ribosome and it is the EFG that pushes RRF during their concerted action for ribosomal subunit dissociation [13, 14]. Thus RRF and unfolded protein access their respective binding sites on ribosome through different routes.…”

“…In presence of EFG-GMPPNP, the unfolded protein failed to split the 70S-tRNA complex (Fig 4B), similarly as RRF is unable to dissociate 70S-tRNA with EFG-GMPPNP and IF3 (Fig 4A). Presumably an EFG-GTP mediated structural transition of 70S-tRNA complex leading to dissociation of tRNA [13, 14] is necessary for RRF or unfolded protein to split 70S.…”

“…EFG-GTP to EFG-GDP transformation is associated with a structural transition of 70S—a prerequisite for tRNA dissociation. In a recent cryo-EM report [14] it has been shown that RRF promotes the rotated state [39]of ribosome and destabilizes the H69 and H44, thereby EFG-GTP facilitates RRF reorientation and actively splits the B2a bridge connecting 50S and 30S (Fig 6). Thus a common aspect of EFG function at the molecular level has been reflected in both elongation and ribosome recycling phases of translation.…”

“…Nucleotides of 23SrRNA interacting with that nascent protein are present in the close proximity of conserved inter-subunit bridge B2a/B2b joining the 50S and 30S [12]. The fact that RRF also interact at the same site [13, 14]may indicate that access of those nucleotides for RRF is only possible once nascent protein leaves the 70S; however isolation of predominant 50S population bound to full length nascent protein in fact lead us to question its role together with that of RRF in dissociating the 70S ribosome.…”

A pseudouridylation switch in rRNA is implicated in ribosome function during the life cycle of Trypanosoma brucei

“…6a ), thus forming an important ribosomal inter-subunit bridge (bridge 2a). H69 is also known to be involved in ribosome recycling 30 31 . Hyper-pseudouridylations occur on H39 (one U), helix H89 (four Us), H90 (one U) and H92 (two Us).…”