Thorsten Mielke

Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA hybrid sites

The elongation cycle of protein synthesis involves the delivery of aminoacyl-transfer RNAs to the aminoacyl-tRNA-binding site (A site) of the ribosome, followed by peptide-bond formation and translocation of the tRNAs through the ribosome to reopen the A site. The translocation reaction is catalysed by elongation factor G (EF-G) in a GTP-dependent manner. Despite the availability of structures of various EF-G–ribosome complexes, the precise mechanism by which tRNAs move through the ribosome still remains unclear. Here we use multiparticle cryoelectron microscopy analysis to resolve two previously unseen subpopulations within Thermus thermophilus EF-G–ribosome complexes at subnanometre resolution, one of them with a partly translocated tRNA. Comparison of these substates reveals that translocation of tRNA on the 30S subunit parallels the swivelling of the 30S head and is coupled to unratcheting of the 30S body. Because the tRNA maintains contact with the peptidyl-tRNA-binding site (P site) on the 30S head and simultaneously establishes interaction with the exit site (E site) on the 30S platform, a novel intra-subunit ‘pe/E’ hybrid state is formed. This state is stabilized by domain IV of EF-G, which interacts with the swivelled 30S-head conformation. These findings provide direct structural and mechanistic insight into the ‘missing link’ in terms of tRNA intermediates involved in the universally conserved translocation process.

Structural Insight Into Nascent Polypeptide Chain-Mediated Translational Stalling

Nascent Chains Revealed Detailed analysis of protein translation and translocation across membranes requires the identification and structural analysis of intermediates involved in these processes (see the Perspective by Kampmann and Blobel). Seidelt et al. (p. 1412, published online 29 October) report the visualization by cryo-electron microscopy of a nascent polypeptide chain in the tunnel of the ribosome at 5.8 angstroms. This resolution allows analysis of the conformation and distinct contacts of the nascent chain within the ribosomal tunnel, which suggests a mechanism by which translational stalling is induced by this peptide. Protein translocation across cellular membranes involves the Sec61 protein, a component of a protein-conducting channel. Whether Sec61 acts as a monomer or as an oligomer during protein translocation has been unclear. Becker et al. (p. 1369, published online 29 October) describe active yeast and mammalian ribosome-Sec61 structures that show the Sec61 complex interacting with the ribosome and a nascent secretory protein signal sequence. The analysis unambiguously reveals that the active protein-conducting channel is a single Sec61 copy with its central pore serving as conduit for the nascent polypeptide. Individual polypeptide nascent chains can adopt distinct conformations within the ribosome exit tunnel. Expression of the Escherichia coli tryptophanase operon depends on ribosome stalling during translation of the upstream TnaC leader peptide, a process for which interactions between the TnaC nascent chain and the ribosomal exit tunnel are critical. We determined a 5.8 angstrom–resolution cryo–electron microscopy and single-particle reconstruction of a ribosome stalled during translation of the tnaC leader gene. The nascent chain was extended within the exit tunnel, making contacts with ribosomal components at distinct sites. Upon stalling, two conserved residues within the peptidyltransferase center adopted conformations that preclude binding of release factors. We propose a model whereby interactions within the tunnel are relayed to the peptidyltransferase center to inhibit translation. Moreover, we show that nascent chains adopt distinct conformations within the ribosomal exit tunnel.

Structure of Monomeric Yeast and Mammalian Sec61 Complexes Interacting with the Translating Ribosome

Nascent Chains Revealed Detailed analysis of protein translation and translocation across membranes requires the identification and structural analysis of intermediates involved in these processes (see the Perspective by Kampmann and Blobel). Seidelt et al. (p. 1412, published online 29 October) report the visualization by cryo-electron microscopy of a nascent polypeptide chain in the tunnel of the ribosome at 5.8 angstroms. This resolution allows analysis of the conformation and distinct contacts of the nascent chain within the ribosomal tunnel, which suggests a mechanism by which translational stalling is induced by this peptide. Protein translocation across cellular membranes involves the Sec61 protein, a component of a protein-conducting channel. Whether Sec61 acts as a monomer or as an oligomer during protein translocation has been unclear. Becker et al. (p. 1369, published online 29 October) describe active yeast and mammalian ribosome-Sec61 structures that show the Sec61 complex interacting with the ribosome and a nascent secretory protein signal sequence. The analysis unambiguously reveals that the active protein-conducting channel is a single Sec61 copy with its central pore serving as conduit for the nascent polypeptide. A single copy of a protein-conducting channel molecule provides a conduit for polypeptide translocation across membranes. The trimeric Sec61/SecY complex is a protein-conducting channel (PCC) for secretory and membrane proteins. Although Sec complexes can form oligomers, it has been suggested that a single copy may serve as an active PCC. We determined subnanometer-resolution cryo–electron microscopy structures of eukaryotic ribosome-Sec61 complexes. In combination with biochemical data, we found that in both idle and active states, the Sec complex is not oligomeric and interacts mainly via two cytoplasmic loops with the universal ribosomal adaptor site. In the active state, the ribosomal tunnel and a central pore of the monomeric PCC were occupied by the nascent chain, contacting loop 6 of the Sec complex. This provides a structural basis for the activity of a solitary Sec complex in cotranslational protein translocation.

Cryo-EM structure of the ribosome-SecYE complex in the membrane environment.

The ubiquitous SecY–Sec61 complex translocates nascent secretory proteins across cellular membranes and integrates membrane proteins into lipid bilayers. Several structures of mostly detergent-solubilized Sec complexes have been reported. Here we present a single-particle cryo-EM structure of the SecYEG complex in a membrane environment, bound to a translating ribosome, at subnanometer resolution. Using the SecYEG complex reconstituted in a so-called Nanodisc, we could trace the nascent polypeptide chain from the peptidyltransferase center into the membrane. The reconstruction allowed for the identification of ribosome–lipid interactions. The rRNA helix 59 (H59) directly contacts the lipid surface and appears to modulate the membrane in immediate vicinity to the proposed lateral gate of the protein-conducting channel (PCC). On the basis of our map and molecular dynamics simulations, we present a model of a signal anchor–gated PCC in the membrane.

GTPase activation of elongation factor EF-Tu by the ribosome during decoding

We have used single‐particle reconstruction in cryo‐electron microscopy to determine a structure of the Thermus thermophilus ribosome in which the ternary complex of elongation factor Tu (EF‐Tu), tRNA and guanine nucleotide has been trapped on the ribosome using the antibiotic kirromycin. This represents the state in the decoding process just after codon recognition by tRNA and the resulting GTP hydrolysis by EF‐Tu, but before the release of EF‐Tu from the ribosome. Progress in sample purification and image processing made it possible to reach a resolution of 6.4 Å. Secondary structure elements in tRNA, EF‐Tu and the ribosome, and even GDP and kirromycin, could all be visualized directly. The structure reveals a complex conformational rearrangement of the tRNA in the A/T state and the interactions with the functionally important switch regions of EF‐Tu crucial to GTP hydrolysis. Thus, the structure provides insights into the molecular mechanism of signalling codon recognition from the decoding centre of the 30S subunit to the GTPase centre of EF‐Tu.

Structure of the ribosome-bound cricket paralysis virus IRES RNA

Internal ribosome entry sites (IRESs) facilitate an alternative, end-independent pathway of translation initiation. A particular family of dicistroviral IRESs can assemble elongation-competent 80S ribosomal complexes in the absence of canonical initiation factors and initiator transfer RNA. We present here a cryo-EM reconstruction of a dicistroviral IRES bound to the 80S ribosome. The resolution of the cryo-EM reconstruction, in the subnanometer range, allowed the molecular structure of the complete IRES in its active, ribosome-bound state to be solved. The structure, harboring three pseudoknot-containing domains, each with a specific functional role, shows how defined elements of the IRES emerge from a compactly folded core and interact with the key ribosomal components that form the A, P and E sites, where tRNAs normally bind. Our results exemplify the molecular strategy for recruitment of an IRES and reveal the dynamic features necessary for internal initiation.

Following the signal sequence from ribosomal tunnel exit to signal recognition particle

Membrane and secretory proteins can be co-translationally inserted into or translocated across the membrane. This process is dependent on signal sequence recognition on the ribosome by the signal recognition particle (SRP), which results in targeting of the ribosome–nascent-chain complex to the protein-conducting channel at the membrane. Here we present an ensemble of structures at subnanometre resolution, revealing the signal sequence both at the ribosomal tunnel exit and in the bacterial and eukaryotic ribosome–SRP complexes. Molecular details of signal sequence interaction in both prokaryotic and eukaryotic complexes were obtained by fitting high-resolution molecular models. The signal sequence is presented at the ribosomal tunnel exit in an exposed position ready for accommodation in the hydrophobic groove of the rearranged SRP54 M domain. Upon ribosome binding, the SRP54 NG domain also undergoes a conformational rearrangement, priming it for the subsequent docking reaction with the NG domain of the SRP receptor. These findings provide the structural basis for improving our understanding of the early steps of co-translational protein sorting.

Cryo-EM structure and rRNA model of a translating eukaryotic 80S ribosome at 5.5-Å resolution

Protein biosynthesis, the translation of the genetic code into polypeptides, occurs on ribonucleoprotein particles called ribosomes. Although X-ray structures of bacterial ribosomes are available, high-resolution structures of eukaryotic 80S ribosomes are lacking. Using cryoelectron microscopy and single-particle reconstruction, we have determined the structure of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution. This map, together with a 6.1-Å map of a Saccharomyces cerevisiae 80S ribosome, has enabled us to model ∼98% of the rRNA. Accurate assignment of the rRNA expansion segments (ES) and variable regions has revealed unique ES–ES and r-protein–ES interactions, providing insight into the structure and evolution of the eukaryotic ribosome.

[alpha]-Helical nascent polypeptide chains visualized within distinct regions of the ribosomal exit tunnel

As translation proceeds, the nascent polypeptide chain passes through a tunnel in the large ribosomal subunit. Although this ribosomal exit tunnel was once thought only to be a passive conduit for the growing nascent chain, accumulating evidence suggests that it may in fact play a more active role in regulating translation and initial protein folding events. Here we have determined single-particle cryo–electron microscopy reconstructions of eukaryotic 80S ribosomes containing nascent chains with high α-helical propensity located within the exit tunnel. The maps enable direct visualization of density for helices as well as allowing the sites of interaction with the tunnel wall components to be elucidated. In particular regions of the tunnel, the nascent chain adopts distinct conformations and establishes specific contacts with tunnel components, both ribosomal RNA and proteins, that have been previously implicated in nascent chain–ribosome interaction.

Coupled Chaperone Action in Folding and Assembly of Hexadecameric Rubisco

Form I Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase), a complex of eight large (RbcL) and eight small (RbcS) subunits, catalyses the fixation of atmospheric CO2 in photosynthesis. The limited catalytic efficiency of Rubisco has sparked extensive efforts to re-engineer the enzyme with the goal of enhancing agricultural productivity. To facilitate such efforts we analysed the formation of cyanobacterial form I Rubisco by in vitro reconstitution and cryo-electron microscopy. We show that RbcL subunit folding by the GroEL/GroES chaperonin is tightly coupled with assembly mediated by the chaperone RbcX2. RbcL monomers remain partially unstable and retain high affinity for GroEL until captured by RbcX2. As revealed by the structure of a RbcL8–(RbcX2)8 assembly intermediate, RbcX2 acts as a molecular staple in stabilizing the RbcL subunits as dimers and facilitates RbcL8 core assembly. Finally, addition of RbcS results in RbcX2 release and holoenzyme formation. Specific assembly chaperones may be required more generally in the formation of complex oligomeric structures when folding is closely coupled to assembly.