ABSTRACT
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.
Subject(s)
Cell Membrane/metabolism , Escherichia coli Proteins/chemistry , Membrane Proteins/chemistry , Ribosomes/chemistry , Cryoelectron Microscopy , Escherichia coli , Escherichia coli Proteins/metabolism , Lipoproteins, HDL/chemistry , Lipoproteins, HDL/metabolism , Membrane Proteins/metabolism , Models, Molecular , Protein Transport , SEC Translocation Channels , Signal Recognition Particle/physiologyABSTRACT
During the process of ribosomal assembly, the essential eukaryotic translation initiation factor 6 (eIF6) is known to act as a ribosomal anti-association factor. However, a molecular understanding of the anti-association activity of eIF6 is still missing. Here we present the cryo-electron microscopy reconstruction of a complex of the large ribosomal subunit with eukaryotic eIF6 from Saccharomyces cerevisiae. The structure reveals that the eIF6 binding site involves mainly rpL23 (L14p in Escherichia coli). Based on our structural data, we propose that the mechanism of the anti-association activity of eIF6 is based on steric hindrance of intersubunit bridge formation around the dynamic bridge B6.
Subject(s)
Peptide Initiation Factors/physiology , Ribosomes/physiology , Saccharomyces cerevisiae Proteins/physiology , Microscopy, Electron , Models, Molecular , Peptide Initiation Factors/chemistry , Protein ConformationABSTRACT
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 alpha-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.
Subject(s)
Peptides/metabolism , Ribosomes/metabolism , Computer Simulation , Cryoelectron Microscopy , Image Processing, Computer-Assisted , Models, Biological , Peptides/chemistry , Protein Biosynthesis , Protein Conformation , Protein Folding , Ribosomes/chemistry , Ribosomes/ultrastructureABSTRACT
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.
Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Protein Biosynthesis , Ribosomes/metabolism , Tryptophanase/genetics , Binding Sites , Cryoelectron Microscopy , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/ultrastructure , Gene Expression Regulation, Bacterial , Image Processing, Computer-Assisted , Models, Biological , Models, Molecular , Operon , Peptidyl Transferases/metabolism , Protein Conformation , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/ultrastructure , Ribosomal Proteins/chemistry , Ribosomal Proteins/metabolism , Ribosomal Proteins/ultrastructure , Ribosomes/ultrastructure , Tryptophanase/biosynthesisABSTRACT
Synthesis of ribosomal RNA (rRNA) by RNA polymerase (Pol) I is the first step in ribosome biogenesis and a regulatory switch in eukaryotic cell growth. Here we report the 12 A cryo-electron microscopic structure for the complete 14-subunit yeast Pol I, a homology model for the core enzyme, and the crystal structure of the subcomplex A14/43. In the resulting hybrid structure of Pol I, A14/43, the clamp, and the dock domain contribute to a unique surface interacting with promoter-specific initiation factors. The Pol I-specific subunits A49 and A34.5 form a heterodimer near the enzyme funnel that acts as a built-in elongation factor and is related to the Pol II-associated factor TFIIF. In contrast to Pol II, Pol I has a strong intrinsic 3'-RNA cleavage activity, which requires the C-terminal domain of subunit A12.2 and, apparently, enables ribosomal RNA proofreading and 3'-end trimming.
Subject(s)
DNA Polymerase I/chemistry , RNA Processing, Post-Transcriptional , RNA, Ribosomal/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/enzymology , Transcription, Genetic , Binding Sites , Cryoelectron Microscopy , Crystallography, X-Ray , DNA Polymerase I/genetics , DNA Polymerase I/metabolism , Models, Molecular , Mutation , Peptide Elongation Factors/chemistry , Peptide Elongation Factors/metabolism , Peptide Initiation Factors/chemistry , Peptide Initiation Factors/metabolism , Promoter Regions, Genetic , Protein Conformation , Protein Interaction Domains and Motifs , Protein Interaction Mapping , Protein Structure, Tertiary , Protein Subunits , RNA, Ribosomal/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Structure-Activity Relationship , Transcription Factors, TFII/chemistry , Transcription Factors, TFII/metabolism , Transcriptional Elongation Factors/chemistry , Transcriptional Elongation Factors/metabolismABSTRACT
Signal sequences of secretory and membrane proteins are recognized by the signal recognition particle (SRP) as they emerge from the ribosome. This results in their targeting to the membrane by docking with the SRP receptor, which facilitates transfer of the ribosome to the translocon. Here, we present the 8 angstrom cryo-electron microscopy structure of a "docking complex" consisting of a SRP-bound 80S ribosome and the SRP receptor. Interaction of the SRP receptor with both SRP and the ribosome rearranged the S domain of SRP such that a ribosomal binding site for the translocon, the L23e/L35 site, became exposed, whereas Alu domain-mediated elongation arrest persisted.
