Ribosomal Exit Tunnel Research Articles

A structural model of the active ribosome-bound membrane protein insertase YidC.

The integration of most membrane proteins into the cytoplasmic membrane of bacteria occurs co-translationally. The universally conserved YidC protein mediates this process either individually as a membrane protein insertase, or in concert with the SecY complex. Here, we present a structural model of YidC based on evolutionary co-variation analysis, lipid-versus-protein-exposure and molecular dynamics simulations. The model suggests a distinctive arrangement of the conserved five transmembrane domains and a helical hairpin between transmembrane segment 2 (TM2) and TM3 on the cytoplasmic membrane surface. The model was used for docking into a cryo-electron microscopy reconstruction of a translating YidC-ribosome complex carrying the YidC substrate FOc. This structure reveals how a single copy of YidC interacts with the ribosome at the ribosomal tunnel exit and identifies a site for membrane protein insertion at the YidC protein-lipid interface. Together, these data suggest a mechanism for the co-translational mode of YidC-mediated membrane protein insertion.

Macrolide antibiotics allosterically predispose the ribosome for translation arrest.

Translation arrest directed by nascent peptides and small cofactors controls expression of important bacterial and eukaryotic genes, including antibiotic resistance genes, activated by binding of macrolide drugs to the ribosome. Previous studies suggested that specific interactions between the nascent peptide and the antibiotic in the ribosomal exit tunnel play a central role in triggering ribosome stalling. However, here we show that macrolides arrest translation of the truncated ErmDL regulatory peptide when the nascent chain is only three amino acids and therefore is too short to be juxtaposed with the antibiotic. Biochemical probing and molecular dynamics simulations of erythromycin-bound ribosomes showed that the antibiotic in the tunnel allosterically alters the properties of the catalytic center, thereby predisposing the ribosome for halting translation of specific sequences. Our findings offer a new view on the role of small cofactors in the mechanism of translation arrest and reveal an allosteric link between the tunnel and the catalytic center of the ribosome.

Identification of a SecM segment required for export-coupled release from elongation arrest.

SecM in Escherichia coli has two functionally crucial regions. The arrest motif near the C-terminus interacts with the ribosomal exit tunnel to arrest its own translational elongation. The signal sequence at the N-terminus directs the SecM nascent polypeptide to the Sec-mediated export pathway to release the arrested state of translation. Here, we addressed the importance of the central region of SecM. Characterization of internal substitution and deletion mutants revealed that a segment from residue 100 to residue 109 is required for the export-coupled release of the SecM nascent chain from the elongation-arrested state. Thus, the central region of SecM is not just a geometric linker but it participates actively in the regulation of translation arrest.

Ribosome profiling reveals sequence-independent post-initiation pausing as a signature of translation.

The journey of a newly synthesized polypeptide starts in the peptidyltransferase center of the ribosome, from where it traverses the exit tunnel. The interior of the ribosome exit tunnel is neither straight nor smooth. How the ribosome dynamics in vivo is influenced by the exit tunnel is poorly understood. Genome-wide ribosome profiling in mammalian cells reveals elevated ribosome density at the start codon and surprisingly the downstream 5th codon position as well. We found that the highly focused ribosomal pausing shortly after initiation is attributed to the geometry of the exit tunnel, as deletion of the loop region from ribosome protein L4 diminishes translational pausing at the 5th codon position. Unexpectedly, the ribosome variant undergoes translational abandonment shortly after initiation, suggesting that there exists an obligatory step between initiation and elongation commitment. We propose that the post-initiation pausing of ribosomes represents an inherent signature of the translation machinery to ensure productive translation.

Sequence-Dependent Elongation Dynamics on Macrolide-Bound Ribosomes

The traditional view of macrolide antibiotics as plugs inside the ribosomal nascent peptide exit tunnel (NPET) has lately been challenged in favor of a more complex, heterogeneous mechanism, where drug-peptide interactions determine the fate of a translating ribosome. To investigate these highly dynamic processes, we applied single-molecule tracking of elongating ribosomes during inhibition of elongation by erythromycin of several nascent chains, including ErmCL and H-NS, which were shown to be, respectively, sensitive and resistant to erythromycin. Peptide sequence-specific changes were observed in translation elongation dynamics in the presence of a macrolide-obstructed NPET. Elongation rates were not severely inhibited in general by the presence of the drug; instead, stalls orpauses were observed as abrupt events. The dynamic pathways of nascent-chain-dependent elongation pausing in the presence of macrolides determine the fate of the translating ribosome stalling or readthrough.

Structural investigation of the folding of an immunoglobulin domain on the ribosome using NMR Spectroscopy (LB197)

Correct protein folding is key to a functional proteome, as globular proteins must acquire their correct three‐dimensional native fold in order to fulfil their biological role. A significant amount of folding occurs co‐translationally on the ribosome and this process is likely to differ from folding of isolated proteins not least through the attachment to the ribosome and the vectorial emergence of the nascent polypeptide from the ribosomal exit tunnel. We are using solution‐state Nuclear Magnetic Resonance (NMR) spectroscopy to study structural aspects of the co‐translational protein folding of stalled ribosome‐bound nascent chain complexes (RNCs) which mimic the progressive emergence of the nascent polypeptide and its acquisition of structure.The current state of our study on the co‐translational folding of a model immunoglobulin domain will be presented including some of the structural models built from chemical shift data of RNCs. These findings contrast with analogous studies of C‐terminal truncations of this domain, indicating some exciting differences between folding on the ribosome and that in bulk solution.[1] Waudby CA, Launay H, Cabrita LD & Christodoulou J. Protein folding on the ribosome studied using NMR spectroscopy. Prog Nucl Magn Reson Spectrosc 74, 57‐75 (2013).

Computational Methods for Measuring the Free Energy of Folding in the Ribosomal Exit Tunnel

As a protein is synthesized in the ribosome, the nascent peptide chain, starting from the peptidyl transferase center (PTC), elongates along the ribosomal exit tunnel, which is ∼10-20A in diameter and ∼100A long. It has been shown that proteins can partially fold inside the exit tunnel and that the ribosome can stabilize the native secondary structure of proteins. However, the mechanism for this stabilization is not yet known at the atomic scale. To determine this mechanism, one can contrast the free energy of α-helix formation in water and in the ribosomal exit tunnel using molecular dynamics (MD) simulations. To determine the free-energy landscapes in water, we employed two computational methods - umbrella sampling (US) and adaptive biasing forces (ABF) - on various polyalanine-containing peptides, using the end-to-end distance of the polyalanine sequence as our reaction coordinate. Since this reaction coordinate does not produce a 1-to-1 correspondence to helical content, successive constraints were added to the simulations, and the changes in the free energy upon addition of each set of constraints were examined. We also applied extended ABF using the helical content of the polyalanine sequence as a reaction and compared the results with the end-to-end distance coordinate. Finally, we used these computational methods to calculate the free-energy landscape along the entire ribosomal exit tunnel with polyalanine-containing sequences placed at different locations.

Interaction of Nascent Chains with the Ribosomal Tunnel Proteins Rpl4, Rpl17, and Rpl39 of Saccharomyces cerevisiae

As translation proceeds, nascent polypeptides pass through an exit tunnel that traverses the large ribosomal subunit. Three ribosomal proteins, termed Rpl4, Rpl17, and Rpl39 expose domains to the interior of the exit tunnel of eukaryotic ribosomes. Here we generated ribosome-bound nascent chains in a homologous yeast translation system to analyze contacts between the tunnel proteins and nascent chains. As model proteins we employed Dap2, which contains a hydrophobic signal anchor (SA) segment, and the chimera Dap2α, in which the SA was replaced with a hydrophilic segment, with the propensity to form an α-helix. Employing a newly developed FLAG exposure assay, we find that the nascent SA segment but not the hydrophilic segment adopted a stable, α-helical structure within the tunnel when the most C-terminal SA residue was separated by 14 residues from the peptidyl transferase center. Using UV cross-linking, antibodies specifically recognizing Rpl17 or Rpl39, and a His6-tagged version of Rpl4, we established that all three tunnel proteins of yeast contact the SA, whereas only Rpl4 and Rpl39 also contact the hydrophilic segment. Consistent with the localization of the tunnel exposed domains of Rpl17 and Rpl39, the SA was in contact with Rpl17 in the middle region and with Rpl39 in the exit region of the tunnel. In contrast, Rpl4 was in contact with nascent chain residues throughout the ribosomal tunnel.

Conjugates of Amino Acids and Peptides with 5-O-Mycaminosyltylonolide and Their Interaction with the Ribosomal Exit Tunnel

During protein synthesis the nascent polypeptide chain (NC) extends through the ribosomal exit tunnel (NPET). Also, the large group of macrolide antibiotics binds in the nascent peptide exit tunnel. In some cases interaction of NC with NPET leads to the ribosome stalling, a significant event in regulation of translation. In other cases NC-ribosome interactions lead to pauses in translation that play an important role in cotranslational folding of polypeptides emerging from the ribosome. The precise mechanism of NC recognition in NPET as well as factors that determine NC conformation in the ribosomal tunnel are unknown. A number of derivatives of the macrolide antibiotic 5-O-mycaminosyltylonolide (OMT) containing N-acylated amino acid or peptide residues were synthesized in order to study potential sites of NC-NPET interactions. The target compounds were prepared by conjugation of protected amino acids and peptides with the C23 hydroxyl group of the macrolide. These OMT derivatives showed high although varying abilities to inhibit the firefly luciferase synthesis in vitro. Three glycil-containing derivatives appeared to be strong inhibitors of translation, more potent than parental OMT. Molecular dynamics (MD) simulation of complexes of tylosin, OMT, and some of OMT derivatives with the large ribosomal subunit of E. coli illuminated a plausible reason for the high inhibitory activity of Boc-Gly-OMT. In addition, the MD study detected a new putative site of interaction of the nascent polypeptide chain with the NPET walls.

Effects of ribosomal exit tunnel on protein's cotranslational folding

In vivo, folding of many proteins occurs during their synthesis in the ribosomeand continues after they have escaped from the ribosomal exit tunnel. Inthis research, we investigate the confinement effects of the ribosome on thecotranslational folding of three proteins, of PDB codes 1PGA, 1CRN and 2RJX,by using a coarse-grained model and molecular dynamics simulation. The exittunnel is modeled as a hollow cylinder attached to a flat wall, whereas aGo-like model is adopted for the proteins. Our results show that theexit tunnel has a strong effect on the folding mechanism by setting an order bywhich the secondary and tertiary structures are formed. For protein 1PGA, thefolding follows two different folding routes. The presence of the tunnel alsoimproves the foldability of protein.

The Bacterial Membrane Insertase YidC Is a Functional Monomer and Binds Ribosomes in a Nascent Chain-Dependent Manner

Thesynthesisandfoldingofproteinsoften occursin kinetically coupled processes in order to avoidthe accumulation of aggregation-prone unfoldedpolypeptide chains. Chaperones are specificallyrecruited to the ribosomal tunnel exit where theywait for the appearance of appropriate binding siteson the emerging polypeptide chains in order toensureearlybindingoffoldingfactors.Foranumberof chaperones and nascent chain modifying en-zymes, the binding sites on the ribosome weremapped to high molecular resolution and theirrelevance for cotranslational protein folding hasbeen studied in detail[1].In contrast, our understanding of how ribosomesassociate with the components that mediate proteininsertionintolipidbilayersisstillrelativelyscarce.Dueto their hydrophobic nature, nascent membraneproteins have a much higher risk to form insolubleaggregates. Hence, a tight coupling of the synthesisand insertion of membrane proteins appears crucial.Two evolutionary conserved systems mediate themembrane integration of proteins: the Sec61/SecYtranslocon (Sec complex) and protein insertases ofthe YidC/Oxa1/Alb3 protein family. The Sec complexforms a translocation pore that allows the completetranslocation of secretory proteins through the mem-brane, as well as the insertion of membrane proteinsviaalateralgate[2].Ribosomescandirectlydockontothe Sec complex so that the ribosomal polypeptidetunnel and the translocation pore in the membraneform one continuous channel [3,4]. The targeting ofribosomes to the Sec complex is thereby initiated bythe signal recognition particle in conjunction with itsreceptor [5].Members of the YidC/Oxa1/Alb3 protein familymediate the insertion of proteins into membranes ofbacteria, mitochondria and chloroplasts, respectively.The precise mode of action is not known, but theirsubstrates typically lack larger soluble domains onthe trans side of the membrane, suggesting thattheir major activity is the integration and folding oftransmembrane segments in the lipid bilayer ratherthanthe translocationof hydrophilic segmentsacrossthe membrane. Nevertheless, the Oxa1 protein of themitochondrialinnermembrane,incooperationwithitshomolog Cox18, was shown to facilitate membranetranslocation of even long hydrophilic domains [6,7]and electrophysiological measurements with purifiedOxa1showedanabilitytoformsubstrate-gatedpores[8].The membrane-embedded insertase domain ofbacterial YidC (Fig. 1a), mitochondrial Oxa1 andchloroplast Alb3 are closely related and functionallyinterchangeable[9–11],butOxa1,incontrasttoYidC,contains a C-terminal ribosome binding domain(Fig. 1b) that connects the protein permanently tomitochondrial ribosomes [12,13]. The permanenttetheringofmitochondrialribosomestothemembraneis presumably a consequence of the evolution of themitochondrial genome during which genes coding forhydrophilic proteins were lost.The bacterial YidC protein lacks a C-terminalribosome binding domain and only exposes a veryshortC-terminalstretchof13residuesintothecytosol.Cross-linking studies indicated that, during the syn-thesis of membrane proteins, YidC and bacterialribosomesareinclosecontact[14,15]butitremainedunclear whether YidC binds bacterial ribosomesdirectly. This important issue was now carefullyaddressed by the group of Arnold Driessen in astudy published in this issue of Journal of MolecularBiology [16]. By use of fluorescence correlationspectroscopy, the authors showed that YidC doesnot associate with bacterial ribosomes (Fig. 1b).Interestingly, the addition of just six histidine residuesto the C-terminus of YidC is sufficient to bind YidC toribosomesparticularlyuponlowpHwheremoreofthehistidineresiduesarepositivelycharged.Thisisavery

Flexibility of the Bacterial Chaperone Trigger Factor in Microsecond-Timescale Molecular Dynamics Simulations

The bacterial chaperone trigger factor (TF) is the first chaperone to be encountered by a nascent protein chain as it emerges from the ribosome exit tunnel. Experimental results suggest that TF possesses considerable conformational flexibility, and in an attempt to provide an atomic-level view of this flexibility, we have performed independent 1.5-μs molecular dynamics simulations of TF in explicit solvent using two different simulation force fields (OPLS-AA/L and AMBER ff99SB-ILDN). Both simulations indicate that TF possesses tremendous flexibility, with huge excursions from the crystallographic conformation caused by reorientations of the protein’s constituent domains; both simulations also predict the formation of extensive contacts between TF’s PPIase domain and the Arm 1 domain that is involved in nascent-chain binding. In the OPLS simulation, however, TF rapidly settles into a very compact conformation that persists for at least 1 μs, whereas in the AMBER simulation, it remains highly dynamic; additional simulations in which the two force fields were swapped suggest that these differences are at least partly attributable to sampling issues. The simulation results provide potential rationalizations of a number of experimental observations regarding TF’s conformational behavior and have implications for using simulations to model TF’s function on translating ribosomes.

Dynamic enzyme docking to the ribosome coordinates N-terminal processing with polypeptide folding

Newly synthesized polypeptides undergo various cotranslational maturation steps, including N-terminal enzymatic processing, chaperone-assisted folding and membrane targeting, but the spatial and temporal coordination of these steps is unclear. We show that Escherichia coli methionine aminopeptidase (MAP) associates with ribosomes through a charged loop that is crucial for nascent-chain processing and cell viability. MAP competes with peptide deformylase (PDF), the first enzyme to act on nascent chains, for binding sites at the ribosomal tunnel exit. PDF has extremely fast association and dissociation kinetics, which allows it to frequently sample ribosomes and ensure the processing of nascent chains after their emergence. Premature recruitment of the chaperone trigger factor, or polypeptide folding, negatively affect processing efficiency. Thus, the fast ribosome association kinetics of PDF and MAP are crucial for the temporal separation of nascent-chain processing from later maturation events, including chaperone recruitment and folding.

On the use of the antibiotic chloramphenicol to target polypeptide chain mimics to the ribosomal exit tunnel

The ribosomal exit tunnel had recently become the centre of many functional and structural studies. Accumulated evidence indicates that the tunnel is not simply a passive conduit for the nascent chain, but a rather functionally important compartment where nascent peptide sequences can interact with the ribosome to signal translation to slow down or even stop. To explore further this interaction, we have synthesized short peptides attached to the amino group of a chloramphenicol (CAM) base, such that when bound to the ribosome these compounds mimic a nascent peptidyl-tRNA chain bound to the A-site of the peptidyltransferase center (PTC). Here we show that these CAM-peptides interact with the PTC of the ribosome while their effectiveness can be modulated by the sequence of the peptide, suggesting a direct interaction of the peptide with the ribosomal tunnel. Indeed, chemical footprinting in the presence of CAM-P2, one of the tested CAM-peptides, reveals protection of 23S rRNA nucleotides located deep within the tunnel, indicating a potential interaction with specific components of the ribosomal tunnel. Collectively, our findings suggest that the CAM-based peptide derivatives will be useful tools for targeting polypeptide chain mimics to the ribosomal tunnel, allowing their conformation and interaction with the ribosomal tunnel to be explored using further biochemical and structural methods.

Electrostatic Effect of the Ribosomal Surface on Nascent Polypeptide Dynamics

The crucial molecular events accompanying protein folding in the cell are still largely unexplored. As nascent polypeptides emerge from the ribosomal exit tunnel, they come in close proximity with the highly negatively charged ribosomal surface. How is the nascent polypeptide influenced by the ribosomal surface? We address this question via the intrinsically disordered protein PIR and a number of its variably charged mutants. Two different populations are identified: one is highly spatially biased, and the other is highly dynamic. The more negatively charged nascent polypeptides emerging from the ribosome are richer in the extremely dynamic population. Hence, nascent proteins with a net negative charge are less likely to interact with the ribosome. Surprisingly, the amplitude of the local motions of the highly dynamic population is much wider than that of disordered polypeptides under physiological conditions, implying that proximity to the ribosomal surface enhances the molecular flexibility of a subpopulation of the nascent protein, much like a denaturing agent would. This effect could be important for a proper structural channeling of the nascent protein and the prevention of cotranslational kinetic trapping. Interestingly, a significant population of the highly spatially biased nascent chain, probably interacting extensively with the ribosome, is present even for very negatively charged nascent proteins. This "sticking" effect likely serves to protect nascent proteins (e.g., from cotranslational aggregation). In all, our results highlight the influence of the ribosome in nascent protein dynamics and show that the ribosome's function in protein biogenesis extends well beyond catalysis of peptide bond formation.

Positively Charged Residues Are the Major Determinants of Ribosomal Velocity

Author SummaryRibosomes do not synthesize protein at a constant rate along transcripts, and changes in translation speed can have knock-on consequences for the expression of that protein, even altering its folding or subcellular localization. It has long been thought that RNA-level features modulate translation rates, whether by delays incurred through the presence of codons that require relatively rare tRNAs, or by regions of mRNA folding that physically impede ribosomal progression. We find on the contrary that it is not RNA-level features but positive charges in the already translated protein that most retard ribosomes, possibly by interacting with the negatively charged ribosomal exit tunnel. We show that positive charge explains the sites where ribosomes stall most commonly within transcripts. We also show why, if protein charge were not considered, one could be misled into suspecting a role for non-optimal codons. Finally, we observe that the poly-A tail provides a massively positively charged terminus no matter in which frame it is translated. A missed stop codon or frameshifting would then lead to a stalled ribosome, which is consistent with experimental data.

Positive Charges Put the Brakes on Ribosomes

The ribosome is at the core of cellular life. It's the incredible machine that trundles along gene transcripts (mRNAs), translating them three letters at a time into the proteins that run most of the workings of the cell. The translation process requires little L-shaped adapters called tRNAs—one end of these recognizes a specific three-letter word (codon) on the mRNA, while the other end carries the corresponding amino acid. The ribosome then glues the amino acids together and shoves the growing protein chain out through an exit tunnel in the back of the ribosome (see Figure 1A). Figure 1 What slows ribosomes down? The freely translating ribosome (A) was thought to be slowed by codons that need rare tRNAs (B) or by mRNA secondary structure (C). Charneski and Hurst, however, show that the dominant braking factor is the presence of positively ... But a question that has occupied many minds over the years is whether the ribosome moves at a more-or-less constant rate along mRNA molecules, or whether its speed is influenced by factors beyond its control. This interest comes from several lines of endeavor. One comprises evolutionary biologists who want to understand the natural optimization of protein translation and the forces that shape it. Another is biotechnologists who aspire to optimize translation speed artificially to maximize protein productivity. The speed of translation is also known to affect the folding and localization of the final protein product—issues of broader physiological impact. Studies on single transcripts seem to show that translation speed is indeed highly variable, and the orthodoxy is that a major determinant of ribosome speed is the abundance of the tRNA molecule needed to interpret each codon. Not all tRNAs are equal, so a codon needing a rare tRNA might slow translation because the ribosome has to wait longer to encounter that tRNA (Figure 1B), and vice versa. Another presumed major player is mRNA secondary structure— if the ribosome needs to plow through a tangled segment of transcript, then it might slow down as energy is expended to unravel the obstacle (Figure 1C). What's changed in the last few years is that rather than relying on data from individual transcripts, the newly developed technique of ribosome profiling gives us a snapshot of where the ribosomes are across all the transcripts in the cell. The resulting “ribosomal occupancy” data can be analyzed to infer a high-resolution pan-transcriptomic map of ribosome speeds that has the potential to tell us exactly what might be slowing ribosomes down. So what is the answer? In this issue of PLOS Biology, Catherine Charneski and Laurence Hurst show that the prime determinant of ribosome speed isn't actually a direct feature of the mRNA itself, but a feature of the emerging protein in the exit tunnel (Figure 1D). Using a previously published dataset, the authors are able to exclude codon/tRNA abundance (Figure 1B) and mRNA secondary structure (Figure 1C) as the most important factors. Ruling out these and all other explanations they could think of, and excluding confounding factors, the authors find out what really seems to matter: if a ribosome has just translated one or more positively charged amino acids (lysine, arginine and, to a lesser extent, histidine), then it slows down. The proposed explanation for this surprisingly simple outcome is that the positive charges interact electrostatically with the negatively charged lining of the ribosomal exit tunnel, gumming it up. The longer the run of positive charges, the greater the effect. The finding that most variation in translation speed could be explained (like romance on the London Underground) largely by opposites attracting in a tunnel challenges the prevailing complex view based on codon usage and mRNA secondary structure; this may well prove controversial, but it brings a provocative clarity to an issue of broad significance. As a tantalizing postscript, the authors also wonder whether the polyA tails that decorate the ends of mRNAs (and which would encode the positively charged amino acid lysine if translated) might've evolved as a sand-trap for runaway ribosomes. Charneski CA, Hurst LD (2013) Positively Charged Residues Are the Major Determinants of Ribosomal Velocity. doi:10.1371/journal.pbio.1001508

Simulation study of the role of the ribosomal exit tunnel on protein folding

To investigate the role of the ribosomal exit tunnel on protein folding, we simulate the initial-stage folding behavior of the protein villin headpiece subdomain HP35 (PDB id: 1yrf) with and without prefolding in the exit tunnel by using an all-atom model and find that prefolding in the exit tunnel could effectively help the protein form native secondary structures. Furthermore, our results show that, after releasing from the exit tunnel, the prefolded chains may have a tendency to form more native contacts than those only in free space and this reduces the conformational space of sampling. Our results may provide an alternative way to explain the fast folding mechanism of proteins in vivo.

Cotranslational Response to Proteotoxic Stress by Elongation Pausing of Ribosomes

Translational control permits cells to respond swiftly to a changing environment. Rapid attenuation of global protein synthesis under stress conditions has been largely ascribed to the inhibition of translation initiation. Here we report that intracellular proteotoxic stress reduces global protein synthesis by halting ribosomes on transcripts during elongation. Deep sequencing of ribosome-protected messenger RNA (mRNA) fragments reveals an early elongation pausing, roughly at the site where nascent polypeptide chains emerge from the ribosomal exit tunnel. Inhibiting endogenous chaperone molecules by a dominant-negative mutant or chemical inhibitors recapitulates the early elongation pausing, suggesting a dual role of molecular chaperones in facilitating polypeptide elongation and cotranslational folding. Our results further support the chaperone "trapping" mechanism in promoting the passage of nascent chains. Our study reveals that translating ribosomes fine tune the elongation rate by sensing the intracellular folding environment. The early elongation pausing represents a cotranslational stress response to maintain the intracellular protein homeostasis.

Structural characterization of a eukaryotic chaperone—the ribosome-associated complex

Ribosome-associated chaperones act in early folding events during protein synthesis. Structural information is available for prokaryotic chaperones (such as trigger factor), but structural understanding of these processes in eukaryotes lags far behind. Here we present structural analyses of the eukaryotic ribosome-associated complex (RAC) from Saccharomyces cerevisiae and Chaetomium thermophilum, consisting of heat-shock protein 70 (Hsp70) Ssz1 and the Hsp40 Zuo1. RAC is an elongated complex that crouches over the ribosomal tunnel exit and seems to be stabilized in a distinct conformation by expansion segment ES27. A unique α-helical domain in Zuo1 mediates ribosome interaction of RAC near the ribosomal proteins L22e and L31e and ribosomal RNA helix H59. The crystal structure of the Ssz1 ATPase domain bound to ATP-Mg²⁺ explains its catalytic inactivity and suggests that Ssz1 may act before the RAC-associated chaperone Ssb. Our study offers insights into the interplay between RAC, the ER membrane-integrated Hsp40-type protein ERj1 and the signal-recognition particle.