Cryo-EM visualization of the ribosome in termination complex with apo-RF3 and RF1

Abstract

Termination of messenger RNA translation in Bacteria and Archaea is initiated by release factors (RFs) 1 or 2 recognizing a stop codon in the ribosomal A site and releasing the peptide from the P-site transfer RNA. After release, RF-dissociation is facilitated by the G-protein RF3. Structures of ribosomal complexes with RF1 or RF2 alone or with RF3 alone—RF3 bound to a non-hydrolyzable GTP-analog—have been reported. Here, we present the cryo-EM structure of a post-termination ribosome containing both apo-RF3 and RF1. The conformation of RF3 is distinct from those of free RF3•GDP and ribosome-bound RF3•GDP(C/N)P. Furthermore, the conformation of RF1 differs from those observed in RF3-lacking ribosomal complexes. Our study provides structural keys to the mechanism of guanine nucleotide exchange on RF3 and to an L12-mediated ribosomal recruitment of RF3. In conjunction with previous observations, our data provide the foundation to structurally characterize the complete action cycle of the G-protein RF3.DOI: http://dx.doi.org/10.7554/eLife.00411.001

RF3:GTP promotes rapid dissociation of the class 1 termination factor

“…3B, lane 8), while His-tagged RF1 binding is substantially diminished in the presence of RF3:GTP and RF3:GDPNP (lanes 12,16). These data are consistent with a model in which the GTPbound form of RF3 stabilizes a conformational state of the ribosome that promotes RF1 dissociation as previously reported (Gao et al 2007;Jin et al 2011;Zhou et al 2012b;Pallesen et al 2013). …”

“…1). Other studies have similarly attempted to remove bound nucleotide (Pallesen et al 2013;Peske et al 2013). Most recently, Wintermeyer and colleagues demonstrated by FPLC the loss of bound nucleotide but found that RF3 activity was then diminished (Peske et al 2013).…”

“…Structural and biochemical studies provide a working model wherein RF3:GTP first binds to RF1-bound ribosomes found in a nonrotated (classical) conformation and then subsequently promotes the transition of the ribosome into the rotated state, with the concomitant dissociation of the class 1 RF (Ermolenko et al 2007;Gao et al 2007;Sternberg et al 2009;Jin et al 2011;Zhou et al 2012b;Pallesen et al 2013). These observations are broadly consistent with the fact that RF3 is closely related to EFG, which can engage ribosomes in a rotated state to facilitate translocation (Ermolenko et al 2007;Pulk and Cate 2013;Tourigny et al 2013;Zhou et al 2013).…”

“…Early structural studies had indicated that RF1/2 and RF3:GTP could not bind simultaneously to the rotated state of the ribosome because of steric clashes between the head of the 30S subunit, the 23S rRNA of the 50S subunit, and the class 1 release factor (Gao et al 2007). Interestingly, a recent cryoEM structure appears to capture a low-occupancy state of the ribosome with both RF3 and RF1 bound (in the nonrotated state); the authors argue that RF3, in this case, has no nucleotide bound (apoRF3) (Pallesen et al 2013). These observations together correlate nicely with the model that had emerged from kinetic studies invoking increased rates of dissociation of RF1/2 when RF3:GTP engages the ribosome following termination.…”

Functional analysis of the uL11 protein impact on translational machinery

“…11,14 Thus, a majority of structural data connect uL11 with the elongation cycle of protein synthesis. However, several structural analyses implicate this protein in interaction with IF2 and RF3, indicating involvement of uL11 in the initiation and termination steps of translation, 15,16 which is also supported by several functional analyses. 17,18 The large ribosomal subunit depleted of this protein is able to bind initiation factor IF2 in vitro but it cannot stimulate GTP hydrolysis required for the subunit joining.…”