The ribosomal tunnel as a functional environment for nascent polypeptide folding and translational stalling

As the nascent polypeptide chain is being synthesized, it passes through a tunnel within the large ribosomal subunit and emerges at the solvent side where protein folding occurs. Despite the universality and conservation of dimensions of the ribosomal tunnel, a functional role for the ribosomal tunnel is only beginning to emerge: Rather than a passive conduit for the nascent chain, accumulating evidence indicates that the tunnel plays a more active role. In this article, we discuss recent structural insights into the role of the tunnel environment, and its implications for protein folding, co-translational targeting and translation regulation.     

The geometry of the ribosomal polypeptide exit tunnel

The geometry of the polypeptide exit tunnel has been determined using the crystal structure of the large ribosomal subunit from Haloarcula marismortui. The tunnel is a component of a much larger, interconnected system of channels accessible to solvent that permeates the subunit and is connected to the exterior at many points. Since water and other small molecules can diffuse into and out of the tunnel along many different trajectories, the large subunit cannot be part of the seal that keeps ions from passing through the ribosome-translocon complex. The structure referred to as the tunnel is the only passage in the solvent channel system that is both large enough to accommodate nascent peptides, and that traverses the particle. For objects of that size, it is effectively an unbranched tube connecting the peptidyl transferase center of the large subunit and the site where nascent peptides emerge. At no point is the tunnel big enough to accommodate folded polypeptides larger than alpha-helices.     

Ribosomal protein eL39 is important for maturation of the nascent polypeptide exit tunnel and proper protein folding during translation

During translation, nascent polypeptide chains travel from the peptidyl transferase center through the nascent polypeptide exit tunnel (NPET) to emerge from 60S subunits. The NPET includes portions of five of the six 25S/5.8S rRNA domains and ribosomal proteins uL4, uL22, and eL39. Internal loops of uL4 and uL22 form the constriction sites of the NPET and are important for both assembly and function of ribosomes. Here, we investigated the roles of eL39 in tunnel construction, 60S biogenesis, and protein synthesis. We show that eL39 is important for proper protein folding during translation. Consistent with a delay in processing of 27S and 7S pre-rRNAs, eL39 functions in pre-60S assembly during middle nucleolar stages. Our biochemical assays suggest the presence of eL39 in particles at these stages, although it is not visualized in them by cryo-electron microscopy. This indicates that eL39 takes part in assembly even when it is not fully accommodated into the body of pre-60S particles. eL39 is also important for later steps of assembly, rotation of the 5S ribonucleoprotein complex, likely through long range rRNA interactions. Finally, our data strongly suggest the presence of alternative pathways of ribosome assembly, previously observed in the biogenesis of bacterial ribosomal subunits.     

Path of nascent polypeptide in exit tunnel revealed by molecular dynamics simulation of ribosome

Molecular dynamics simulations were carried out on Thermus thermophilus 70S ribosome with and without a nascent polypeptide inside the exit tunnel. Modeling of the polypeptide in the tunnel revealed two possible paths: one over Arg⁹² of L22 and one under (from the viewpoint of 50S on top of 30S). A strong interaction between L4 and Arg⁹² was observed without the polypeptide and when it passed over Arg⁹². However, when the polypeptide passed under, Arg⁹² repositioned to interact with Ade²⁰⁵⁹ of 23S rRNA. Using steered molecular dynamics the polypeptide could be pulled through the L4-L22 constriction when situated under Arg⁹², but did not move when over. These results suggest that the tunnel is closed by the Arg⁹²-L4 interaction before elongation of the polypeptide and the tunnel leads the entering polypeptide from the peptidyl transferase center to the passage under Arg⁹², causing Arg⁹² to switch to an open position. It is possible, therefore, that Arg⁹² plays the role of a gate, opening and closing the tunnel at L4-L22. There is some disagreement over whether the tunnel is dynamic or rigid. At least within the timescale of our simulations conformational analysis showed that global motions mainly involve relative movement of the 50S and 30S subunits and seem not to affect the conformation of the tunnel.     

Hydrophobic and electrostatic interactions modulate protein escape at the ribosomal exit tunnel

After translation, nascent proteins must escape the ribosomal exit tunnel to attain complete folding to their native states. This escape process also frees up the ribosome tunnel for a new translation job. In this study, we investigate the impacts of energetic interactions between the ribosomal exit tunnel and nascent proteins on the protein escape process by molecular dynamics simulations using partially coarse-grained models that incorporate hydrophobic and electrostatic interactions of the ribosome tunnel of Haloarcula marismortui with nascent proteins. We find that, in general, attractive interactions slow down the protein escape process, whereas repulsive interactions speed it up. For the small globular proteins considered, the median escape time correlates with both the number of hydrophobic residues, N_h, and the net charge, Q, of a nascent protein. A correlation coefficient exceeding 0.96 is found for the relation between the median escape time and a combined quantity of N_h + 5.9Q, suggesting that it is ∼6 times more efficient to modulate the escape time by changing the total charge than the number of hydrophobic residues. The estimated median escape times are found in the submillisecond-to-millisecond range, indicating that the escape does not delay the ribosome recycling. For various types of the tunnel model, with and without hydrophobic and electrostatic interactions, the escape time distribution always follows a simple diffusion model that describes the escape process as a downhill drift of a Brownian particle, suggesting that nascent proteins escape along barrier-less pathways at the ribosome tunnel.     

Association of nascent polypeptide and transfer ribonucleic acid with 30S ribosomal subunits

1. Crude extracts of Escherichia coli programmed in protein synthesis by endogenous mRNA have incorporated amino acids into protein. Analysis of such extracts by sucrose-gradient centrifugation in low Mg(2+) concentration has revealed that 30S ribosomal subunits carry associated radioactive material of which a considerable proportion can be removed from ribosomes by treatment of pre-labelled extracts with puromycin. 2. Gradient analyses of incorporations carried out in the additional presence of added (32)P-labelled tRNA have indicated that tRNA sediments in the regions of the newly synthesized nascent protein and that both labels are associated with all ribosomal components detected on the gradients under the experimental conditions employed. 3. 30S ribosomal subunits carrying both (32)P and (14)C labels have been isolated, disrupted with sodium dodecyl sulphate, and analysed by chromatography on Sephadex G-200 columns. Both labels elute closely together and well away from a tRNA marker analysed under identical conditions. 4. It is proposed that 30S ribosomal subunits, isolated from extracts which have synthesized nascent peptides under the direction of endogenous mRNA, carry associated peptidyl-tRNA.     

The conformation of a nascent polypeptide inside the ribosome tunnel affects protein targeting and protein folding

In this report, we describe insights into the function of the ribosome tunnel that were obtained through an analysis of an unusual 25 residue N‐terminal motif (EspP_1‐25) associated with the signal peptide of the Escherichia coli EspP protein. It was previously shown that EspP_1‐25 inhibits signal peptide recognition by the signal recognition particle, and we now show that fusion of EspP_1‐25 to a cytoplasmic protein causes it to aggregate. We obtained two lines of evidence that both of these effects are attributable to the conformation of EspP_1‐25 inside the ribosome tunnel. First, we found that mutations in EspP_1‐25 that abolished its effects on protein targeting and protein folding altered the cross‐linking of short nascent chains to ribosomal components. Second, we found that a mutation in L22 that distorts the tunnel mimicked the effects of the EspP_1‐25 mutations on protein biogenesis. Our results provide evidence that the conformation of a polypeptide inside the ribosome tunnel can influence protein folding under physiological conditions and suggest that ribosomal mutations might increase the solubility of at least some aggregation‐prone proteins produced in E. coli.     

Interrelationships between yeast ribosomal protein assembly events and transient ribosome biogenesis factors interactions in early pre-ribosomes

Early steps of eukaryotic ribosome biogenesis require a large set of ribosome biogenesis factors which transiently interact with nascent rRNA precursors (pre-rRNA). Most likely, concomitant with that initial contacts between ribosomal proteins (r-proteins) and ribosome precursors (pre-ribosomes) are established which are converted into robust interactions between pre-rRNA and r-proteins during the course of ribosome maturation. Here we analysed the interrelationship between r-protein assembly events and the transient interactions of ribosome biogenesis factors with early pre-ribosomal intermediates termed 90S pre-ribosomes or small ribosomal subunit (SSU) processome in yeast cells. We observed that components of the SSU processome UTP-A and UTP-B sub-modules were recruited to early pre-ribosomes independently of all tested r-proteins. On the other hand, groups of SSU processome components were identified whose association with early pre-ribosomes was affected by specific r-protein assembly events in the head-platform interface of the SSU. One of these components, Noc4p, appeared to be itself required for robust incorporation of r-proteins into the SSU head domain. Altogether, the data reveal an emerging network of specific interrelationships between local r-protein assembly events and the functional interactions of SSU processome components with early pre-ribosomes. They point towards some of these components being transient primary pre-rRNA in vivo binders and towards a role for others in coordinating the assembly of major SSU domains.     

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.     

Ribosome-associated complex binds to ribosomes in close proximity of Rpl31 at the exit of the polypeptide tunnel in yeast

Ribosome-associated complex (RAC) consists of the Hsp40 homolog Zuo1 and the Hsp70 homolog Ssz1. The chaperone participates in the biogenesis of newly synthesized polypeptides. Here we have identified yeast Rpl31, a component of the large ribosomal subunit, as a contact point of RAC at the polypeptide tunnel exit. Rpl31 is encoded by RPL31a and RPL31b, two closely related genes. Δrpl31aΔrpl31b displayed slow growth and sensitivity to low as well as high temperatures. In addition, Δrpl31aΔrpl31b was highly sensitive toward aminoglycoside antibiotics and suffered from defects in translational fidelity. With the exception of sensitivity at elevated temperature, the phenotype resembled yeast strains lacking one of the RAC subunits or Rpl39, another protein localized at the tunnel exit. Defects of Δrpl31aΔrpl31bΔzuo1 did not exceed that of Δrpl31aΔrpl31b or Δzuo1. However, the combined deletion of RPL31a, RPL31b, and RPL39 was lethal. Moreover, RPL39 was a multicopy suppressor, whereas overexpression of RAC failed to rescue growth defects of Δrpl31aΔrpl31b. The findings are consistent with a model in that Rpl31 and Rpl39 independently affect a common ribosome function, whereas Rpl31 and RAC are functionally interdependent. Rpl31, while not essential for binding of RAC to the ribosome, might be involved in proper function of the chaperone complex.     

Analysis of the interplay of protein biogenesis factors at the ribosome exit site reveals new role for NAC

The ribosome exit site is a focal point for the interaction of protein-biogenesis factors that guide the fate of nascent polypeptides. These factors include chaperones such as NAC, N-terminal-modifying enzymes like Methionine aminopeptidase (MetAP), and the signal recognition particle (SRP), which targets secretory and membrane proteins to the ER. These factors potentially compete with one another in the short time-window when the nascent chain first emerges at the exit site, suggesting a need for regulation. Here, we show that MetAP contacts the ribosome at the universal adaptor site where it is adjacent to the α subunit of NAC. SRP is also known to contact the ribosome at this site. In the absence of NAC, MetAP and SRP antagonize each other, indicating a novel role for NAC in regulating the access of MetAP and SRP to the ribosome. NAC also functions in SRP-dependent targeting and helps to protect substrates from aggregation before translocation.     

S-adenosyl-L-methionine induces compaction of nascent peptide chain inside the ribosomal exit tunnel upon translation arrest in the Arabidopsis CGS1 gene

Expression of the Arabidopsis CGS1 gene, encoding the first committed enzyme of methionine biosynthesis, is feedback-regulated in response to S-adenosyl-L-methionine (AdoMet) at the mRNA level. This regulation is first preceded by temporal arrest of CGS1 translation elongation at the Ser-94 codon. AdoMet is specifically required for this translation arrest, although the mechanism by which AdoMet acts with the CGS1 nascent peptide remained elusive. We report here that the nascent peptide of CGS1 is induced to form a compact conformation within the exit tunnel of the arrested ribosome in an AdoMet-dependent manner. Cysteine residues introduced into CGS1 nascent peptide showed reduced ability to react with polyethyleneglycol maleimide in the presence of AdoMet, consistent with a shift into the ribosomal exit tunnel. Methylation protection and UV cross-link assays of 28 S rRNA revealed that induced compaction of nascent peptide is associated with specific changes in methylation protection and UV cross-link patterns in the exit tunnel wall. A 14-residue stretch of amino acid sequence, termed the MTO1 region, has been shown to act in cis for CGS1 translation arrest and mRNA degradation. This regulation is lost in the presence of mto1 mutations, which cause single amino acid alterations within MTO1. In this study, both the induced peptide compaction and exit tunnel change were found to be disrupted by mto1 mutations. These results suggest that the MTO1 region participates in the AdoMet-induced arrest of CGS1 translation by mediating changes of the nascent peptide and the exit tunnel wall.     

The central core region of yeast ribosomal protein L11 is important for subunit joining and translational fidelity

Yeast ribosomal protein L11 is positioned at the intersubunit cleft of the large subunit central protuberance, forming an intersubunit bridge with the small subunit protein S18. Mutants were engineered in the central core region of L11 which interacts with Helix 84 of the 25S rRNA. Numerous mutants in this region conferred 60S subunit biogenesis defects. Specifically, many mutations of F96 and the A66D mutant promoted formation of halfmers as assayed by sucrose density ultracentrifugation. Halfmer formation was not due to deficiency in 60S subunit production, suggesting that the mutants affected subunit-joining. Chemical modification analyses indicated that the A66D mutant, but not the F96 mutants, promoted changes in 25S rRNA structure, suggesting at least two modalities for subunit joining defects. 25S rRNA structural changes were located both adjacent to A66D (in H84), and more distant (in H96-7). While none of the mutants significantly affected ribosome/tRNA binding constants, they did have strong effects on cellular growth at both high and low temperatures, in the presence of translational inhibitors, and promoted changes in translational fidelity. Two distinct mechanisms are proposed by which L11 mutants may affect subunit joining, and identification of the amino acids associated with each of these processes are presented. These findings may have implications for our understanding of multifaceted diseases such as Diamond–Blackfan anemia which have been linked in part with mutations in L11.     

The ribosomal protein Rps15p is required for nuclear exit of the 40S subunit precursors in yeast

We have conducted a genetic screen in order to identify ribosomal proteins of Saccharomyces cerevisiae involved in nuclear export of the small subunit precursors. This has led us to distinguish Rps15p as a protein dispensable for maturation of the pre-40S particles, but whose assembly into the pre-ribosomes is a prerequisite to their nuclear exit. Upon depletion of Rps15p, 20S pre-rRNA is released from the nucleolus and retained in the nucleus, without alteration of the pre-rRNA early cleavages. In contrast, Rps18p, which contacts Rps15p in the small subunit, is required upstream for pre-rRNA processing at site A2. Most pre-40S specific factors are correctly associated with the intermediate particles accumulating in the nucleus upon Rps15p depletion, except the late-binding proteins Tsr1p and Rio2p. Here we show that these two proteins are dispensable for nuclear exit; instead, they participate in 20S pre-rRNA processing in the cytoplasm. We conclude that, during the final maturation steps in the nucleus, incorporation of the ribosomal protein Rps15p is specifically required to render the pre-40S particles competent for translocation to the cytoplasm.     

In the Archaea Haloferax volcanii, membrane protein biogenesis and protein synthesis rates are affected by decreased ribosomal binding to the translocon

In the haloarchaea Haloferax volcanii, ribosomes are found in the cytoplasm and membrane-bound at similar levels. Transformation of H. volcanii to express chimeras of the translocon components SecY and SecE fused to a cellulose-binding domain substantially decreased ribosomal membrane binding, relative to non-transformed cells, likely due to steric hindrance by the cellulose-binding domain. Treatment of cells with the polypeptide synthesis terminator puromycin, with or without low salt washes previously shown to prevent in vitro ribosomal membrane binding in halophilic archaea, did not lead to release of translocon-bound ribosomes, indicating that ribosome release is not directly related to the translation status of a given ribosome. Release was, however, achieved during cell starvation or stationary growth, pointing at a regulated manner of ribosomal release in H. volcanii. Decreased ribosomal binding selectively affected membrane protein levels, suggesting that membrane insertion occurs co-translationally in Archaea. In the presence of chimera-incorporating sterically hindered translocons, the reduced ability of ribosomes to bind in the transformed cells modulated protein synthesis rates over time, suggesting that these cells manage to compensate for the reduction in ribosome binding. Possible strategies for this compensation, such as a shift to a post-translational mode of membrane protein insertion or maintained ribosomal membrane-binding, are discussed.     

Mechanism of regulating the expression of λN gene by ribosomal protein at translational level

In ribosomal protein S12 mutant or L24 mutant the expression of λN gene was depressed at translational level. To study its mechanism the λN gene region of λN -lacZ gene fusion was trimmed from its 5′ end to 3′ end with DNA exonuclease III (DNA cxoIII) in order to alter the TIR (translational initiation region) and the ding region of λN gene. After DNA sequencing 23 species of different λN-lacZ fused genes were obtained. The β-galactosidase activities of these deletants in ribosomal protein mutant were compared with that in wild type strain. The result indicated that (i) S12 mutant could affect 305 subunit’s binding to the TIR of λN gene messenger and cause the difficulty in forming 30s initiation complex and then decrease the efficiency of translational initiation; (ii) in S12 mutant the coding region of λN gene alw affected the expression λN gene; (iii) in L24 mutant the inhibition of λN gene expression was not related to translational initiation and the 5′ end of the coding region of λN gene, but related to the 3′ end of λN gene.     

The polypeptide tunnel exit of the mitochondrial ribosome is tailored to meet the specific requirements of the organelle

The ribosomal polypeptide tunnel exit is the site where a variety of factors interact with newly synthesized proteins to guide them through the early steps of their biogenesis. In mitochondrial ribosomes, this site has been considerably modified in the course of evolution. In contrast to all other translation systems, mitochondrial ribosomes are responsible for the synthesis of only a few hydrophobic membrane proteins that are essential subunits of the mitochondrial respiratory chain. Membrane insertion of these proteins occurs co‐translationally and is connected to a sophisticated assembly process that not only includes the assembly of the different subunits but also the acquisition of redox co‐factors. Here, we describe how mitochondrial translation is organized in the context of respiratory chain assembly and speculate how alteration of the ribosomal tunnel exit might allow the establishment of a subset of specialized ribosomes that individually organize the early steps in the biogenesis of distinct mitochondrially‐encoded proteins.     

Mechanism of regulating the expression of λN gene by ribosomal protein at translational level

In ribosomal protein S12 mutant or L24 mutant the expression of λN gene was depressed at translational level. To study its mechanism the λN gene region of λN -lacZ gene fusion was trimmed from its 5′ end to 3′ end with DNA exonuclease III (DNA cxoIII) in order to alter the TIR (translational initiation region) and the ding region of λN gene. After DNA sequencing 23 species of different λN-lacZ fused genes were obtained. The β-galactosidase activities of these deletants in ribosomal protein mutant were compared with that in wild type strain. The result indicated that (i) S12 mutant could affect 305 subunit’s binding to the TIR of λN gene messenger and cause the difficulty in forming 30s initiation complex and then decrease the efficiency of translational initiation; (ii) in S12 mutant the coding region of λN gene alw affected the expression λN gene; (iii) in L24 mutant the inhibition of λN gene expression was not related to translational initiation and the 5′ end of the coding region of λN gene, but related to the 3′ end of λN gene. Project supported by the National Natural Science Foundation of China (Grant Nos. 39480014, 39570162) and Chinese Academy of Sciences.