Structures of the E. coli translating ribosome with SRP and its receptor and with the translocon

Abstract

Co-translational protein targeting to membranes is a universally conserved process. Central steps include cargo recognition by the signal recognition particle and handover to the Sec translocon. Here we present snapshots of key co-translational-targeting complexes solved by cryo-electron microscopy at near-atomic resolution, establishing the molecular contacts between the Escherichia coli translating ribosome, the signal recognition particle and the translocon. Our results reveal the conformational changes that regulate the latching of the signal sequence, the release of the heterodimeric domains of the signal recognition particle and its receptor, and the handover of the signal sequence to the translocon. We also observe that the signal recognition particle and the translocon insert-specific structural elements into the ribosomal tunnel to remodel it, possibly to sense nascent chains. Our work provides structural evidence for a conformational state of the signal recognition particle and its receptor primed for translocon binding to the ribosome–nascent chain complex.

The ribosome and its role in protein folding: looking through a magnifying glass

“…The SRP is another protein which competes for the NC via the uL23 binding site (Schibich et al, 2016) and recognizes the N-terminal signal peptide sequence on NCs to initiate protein translocation. Recent cryo-EM analysis of the 70S-SRP complex have captured intermediate states of SRP bound to the 70S ribosome, providing snapshots of SRP engagement with the ribosome-emerging NC (Jomaa et al, 2016). This occurs in a co-translational manner, before targeting the NC to the membrane compartment of the cell (von Loeffelholz et al, 2015;Jomaa et al, 2016;).…”

“…Recent cryo-EM analysis of the 70S-SRP complex have captured intermediate states of SRP bound to the 70S ribosome, providing snapshots of SRP engagement with the ribosome-emerging NC (Jomaa et al, 2016). This occurs in a co-translational manner, before targeting the NC to the membrane compartment of the cell (von Loeffelholz et al, 2015;Jomaa et al, 2016;). These recent accomplishments in the structural analysis of dynamic ribosome complexes demonstrate how increasingly complex questions related to ribosome function are no longer beyond the reach of structural biology.…”

Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery

“…Continued advances in single-particle cryo-EM over the next two decades enabled resolution analysis of non-crystalline samples with high internal symmetry such as icosahedral and helical viruses (Ge and Zhou, 2011; Settembre et al, 2011; Yu et al, 2008; Zhang et al, 2010). Large and relatively stable complexes such as ribosomes also proved especially amenable to analysis using cryo-EM methods, first at medium resolution (Matadeen et al, 1999; Rawat et al, 2003) and more recently at near-atomic resolution (Amunts et al, 2014; Fischer et al, 2015; Jomaa et al, 2016; Wong et al, 2014). These successes have now been extended to a wide spectrum of protein complexes, including several integral membrane proteins (Bai et al, 2015b; Du et al, 2015; Liao et al, 2013; Matthies et al, 2016).…”

Driving Forces of Translocation Through Bacterial Translocon SecYEG

“…Another engaged structure proposes even greater squeezing of the “ring” when compared to the ellipse of the resting state, with simultaneous tightening of the lateral gate (pdb 5GAE (Jomaa et al 2016)). How (a) substrate sampling between the aqueous environment of the channel and the hydrophobic membrane interior or (b) the translocation of co-translational substrates may occur under these conditions has remained unclear.…”

“…In contrast to most of the structures with a translocation intermediate such as 3J46 (Park et al 2013), 5GAE (Jomaa et al 2016), and 5EUL (Li et al 2016), the reconstituted SecY-signal peptide, SecY-proOmpA complex, or SecY-ribosome complex appear to be ion permeable at small transmembrane potentials (Knyazev et al 2013; Knyazev et al 2014). The complexes exclude ions when physiological values of membrane potential (Fig.…”