Mechanisms of SecM-Mediated Stalling in the Ribosome

Nascent-peptide modulation of translation is a common regulatory mechanism of gene expression. In this mechanism, while the nascent peptide is still in the exit tunnel of the ribosome, it induces translational pausing, thereby controlling the expression of downstream genes. One example is SecM, which inhibits peptide-bond formation in the ribosome's peptidyl transferase center (PTC) during its own translation, upregulating the expression of the protein translocase SecA. Although biochemical experiments and cryo-electron microscopy data have led to the identification of some residues involved in SecM recognition, the full pathway of interacting residues that connect SecM to the PTC through the ribosome has not yet been conclusively established. Here, using the cryo-electron microscopy data, we derived the first (to our knowledge) atomic model of the SecM-stalled ribosome via molecular-dynamics flexible fitting, complete with P- and A-site tRNAs. Subsequently, we carried out simulations of native and mutated SecM-stalled ribosomes to investigate possible interaction pathways between a critical SecM residue, R163, and the PTC. In particular, the simulations reveal the role of SecM in altering the position of the tRNAs in the ribosome, and thus demonstrate how the presence of SecM in the exit tunnel induces stalling. Finally, steered molecular-dynamics simulations in which SecM was pulled toward the tunnel exit suggest how SecA interacting with SecM from outside the ribosome relieves stalling.     

The ribosomal exit tunnel functions as a discriminating gate

Translation of SecM stalls unless its N-terminal part is "pulled" by the protein export machinery. Here we show that the sequence motif FXXXXWIXXXXGIRAGP that includes a specific arrest point (Pro) causes elongation arrest within the ribosome. Mutations that bypass the elongation arrest were isolated in 23S rRNA and L22 r protein. Such suppressor mutations occurred at a few specific residues of these components, which all face the narrowest constriction of the ribosomal exit tunnel. Thus, we suggest that this region of the exit tunnel interacts with nascent translation products and functions as a discriminating gate.     

Genetic Identification of Nascent Peptides That Induce Ribosome Stalling

Several nascent peptides stall ribosomes during their own translation in both prokaryotes and eukaryotes. Leader peptides that induce stalling can regulate downstream gene expression. Interestingly, stalling peptides show little sequence similarity and interact with the ribosome through distinct mechanisms. To explore the scope of regulation by stalling peptides and to better understand the mechanism of stalling, we identified and characterized new examples from random libraries. We created a genetic selection that ties the life of Escherichia coli cells to stalling at a specific site. This selection relies on the natural bacterial system that rescues arrested ribosomes. We altered transfer-messenger RNA, a key component of this rescue system, to direct the completion of a necessary protein if and only if stalling occurs. We identified three classes of stalling peptides: C-terminal Pro residues, SecM-like peptides, and the novel stalling sequence FXXYXIWPP. Like the leader peptides SecM and TnaC, the FXXYXIWPP peptide induces stalling efficiently by inhibiting peptidyl transfer. The nascent peptide exit tunnel and peptidyltransferase center are implicated in this stalling event, although mutations in the ribosome affect stalling on SecM and FXXYXIWPP differently. We conclude that ribosome stalling can be caused by numerous sequences and is more common than previously believed.     

Electrostatics in the ribosomal tunnel modulate chain elongation rates

Electrostatic potentials along the ribosomal exit tunnel are nonuniform and negative. The significance of electrostatics in the tunnel remains relatively uninvestigated, yet they are likely to play a role in translation and secondary folding of nascent peptides. To probe the role of nascent peptide charges in ribosome function, we used a molecular tape measure that was engineered to contain different numbers of charged amino acids localized to known regions of the tunnel and measured chain elongation rates. Positively charged arginine or lysine sequences produce transient arrest (pausing) before the nascent peptide is fully elongated. The rate of conversion from transiently arrested to full-length nascent peptide is faster for peptides containing neutral or negatively charged residues than for those containing positively charged residues. We provide experimental evidence that extraribosomal mechanisms do not account for this charge-specific pausing. We conclude that pausing is due to charge-specific interactions between the tunnel and the nascent peptide.     

Elongation arrest by SecM via a cascade of ribosomal RNA rearrangements

In E. coli, the SecM nascent polypeptide causes elongation arrest, while interacting with 23S RNA bases A2058 and A749-753 in the exit tunnel of the large ribosomal subunit. We compared atomic models fitted by real-space refinement into cryo-electron microscopy reconstructions of a pretranslocational and SecM-stalled E. coli ribosome complex. A cascade of RNA rearrangements propagates from the exit tunnel throughout the large subunit, affecting intersubunit bridges and tRNA positions, which in turn reorient small subunit RNA elements. Elongation arrest could result from the inhibition of mRNA.(tRNAs) translocation, E site tRNA egress, and perhaps translation factor activation at the GTPase-associated center. Our study suggests that the specific secondary and tertiary arrangement of ribosomal RNA provides the basis for internal signal transduction within the ribosome. Thus, the ribosome may itself have the ability to regulate its progression through translation by modulating its structure and consequently its receptivity to activation by cofactors.     

Statics of the Ribosomal Exit Tunnel: Implications for Cotranslational Peptide Folding, Elongation Regulation, and Antibiotics Binding

A sophisticated interplay between the static properties of the ribosomal exit tunnel and its functional role in cotranslational processes is revealed by constraint counting on topological network representations of large ribosomal subunits from four different organisms. As for the global flexibility characteristics of the subunit, the results demonstrate a conserved stable structural environment of the tunnel. The findings render unlikely that deformations of the tunnel move peptides down the tunnel in an active manner. Furthermore, the stable environment rules out that the tunnel can adapt widely so as to allow tertiary folding of nascent chains. Nevertheless, there are local zones of flexible nucleotides within the tunnel, between the peptidyl transferase center and the tunnel constriction, and at the tunnel exit. These flexible zones strikingly agree with previously identified folding zones. As for cotranslational elongation regulation, flexible residues in the β-hairpin of the ribosomal L22 protein were verified, as suggested previously based on structural results. These results support the hypothesis that L22 can undergo conformational changes that regulate the tunnel voyage of nascent polypeptides. Furthermore, rRNA elements, for which conformational changes have been observed upon interaction of the tunnel wall with a nascent SecM peptide, are less strongly coupled to the subunit core. Sequences of coupled rigid clusters are identified between the tunnel and some of these elements, suggesting signal transmission by a domino-like mechanical coupling. Finally, differences in the flexibility of the glycosidic bonds of bases that form antibiotics-binding crevices within the peptidyl transferase center and the tunnel region are revealed for ribosomal structures from different kingdoms. In order to explain antibiotics selectivity, action, and resistance, according to these results, differences in the degrees of freedom of the binding regions may need to be considered.     

SecM-stalled ribosomes adopt an altered geometry at the peptidyl transferase center

As nascent polypeptide chains are synthesized, they pass through a tunnel in the large ribosomal subunit. Interaction between specific nascent chains and the ribosomal tunnel is used to induce translational stalling for the regulation of gene expression. One well-characterized example is the Escherichia coli SecM (secretion monitor) gene product, which induces stalling to up-regulate translation initiation of the downstream secA gene, which is needed for protein export. Although many of the key components of SecM and the ribosomal tunnel have been identified, understanding of the mechanism by which the peptidyl transferase center of the ribosome is inactivated has been lacking. Here we present a cryo-electron microscopy reconstruction of a SecM-stalled ribosome nascent chain complex at 5.6 Å. While no cascade of rRNA conformational changes is evident, this structure reveals the direct interaction between critical residues of SecM and the ribosomal tunnel. Moreover, a shift in the position of the tRNA–nascent peptide linkage of the SecM-tRNA provides a rationale for peptidyl transferase center silencing, conditional on the simultaneous presence of a Pro-tRNA^Pro in the ribosomal A-site. These results suggest a distinct allosteric mechanism of regulating translational elongation by the SecM stalling peptide.     

Early encounters of a nascent membrane protein: specificity and timing of contacts inside and outside the ribosome

An unbiased photo-cross-linking approach was used to probe the "molecular path" of a growing nascent Escherichia coli inner membrane protein (IMP) from the peptidyl transferase center to the surface of the ribosome. The nascent chain was initially in proximity to the ribosomal proteins L4 and L22 and subsequently contacted L23, which is indicative of progression through the ribosome via the main ribosomal tunnel. The signal recognition particle (SRP) started to interact with the nascent IMP and to target the ribosome-nascent chain complex to the Sec-YidC complex in the inner membrane when maximally half of the transmembrane domain (TM) was exposed from the ribosomal exit. The combined data suggest a flexible tunnel that may accommodate partially folded nascent proteins and parts of the SRP and SecY. Intraribosomal contacts of the nascent chain were not influenced by the presence of a functional TM in the ribosome.     

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.     

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.     

Nascent peptide in the "birth canal" of the ribosome

All proteins assembled by the ribosome must pass through the nascent-peptide exit tunnel. Some nascent peptides can specifically interact with the tunnel and affect the functions of the ribosome. The molecular mechanisms of such interactions and of the ribosome response are currently unknown. However, a recent study has revealed elements of the tunnel that might be involved in sensing the nascent-peptide sequence.     

Nascent SecM Chain Outside the Ribosome Reinforces Translation Arrest

SecM, a bacterial secretion monitor protein, contains a specific amino acid sequence at its C-terminus, called arrest sequence, which interacts with the ribosomal tunnel and arrests its own translation. The arrest sequence is sufficient and necessary for stable translation arrest. However, some previous studies have suggested that the nascent chain outside the ribosome affects the stability of translation arrest. To clarify this issue, we performed in vitro translation assays with HaloTag proteins fused to the C-terminal fragment of E. coli SecM containing the arrest sequence or the full-length SecM. We showed that the translation of HaloTag proteins, which are fused to the fragment, is not effectively arrested, whereas the translation of HaloTag protein fused to full-length SecM is arrested efficiently. In addition, we observed that the nascent SecM chain outside the ribosome markedly stabilizes the translation arrest. These results indicate that changes in the nascent polypeptide chain outside the ribosome can affect the stability of translation arrest; the nascent SecM chain outside the ribosome stabilizes the translation arrest.     

The extended loops of ribosomal proteins L4 and L22 are not required for ribosome assembly or L4-mediated autogenous control

Ribosomal proteins L4 and L22 both have a globular domain that sits on the surface of the large ribosomal subunit and an extended loop that penetrates its core. The tips of both loops contribute to the lining of the peptide exit tunnel and have been implicated in a gating mechanism that might regulate the exit of nascent peptides. Also, the extensions of L4 and L22 contact multiple domains of 23S rRNA, suggesting they might facilitate rRNA folding during ribosome assembly. To learn more about the roles of these extensions, we constructed derivatives of both proteins that lack most of their extended loops. Our analysis of ribosomes carrying L4 or L22 deletion proteins did not detect any significant difference in their sedimentation property or polysome distribution. Also, the role of L4 in autogenous control was not affected. We conclude that these extensions are not required for ribosome assembly or for L4-mediated autogenous control of the S10 operon.     

Homogeneous stalled ribosome nascent chain complexes produced in vivo or in vitro

Cotranslational protein maturation is often studied in cell-free translation mixtures, using stalled ribosome-nascent chain complexes produced by translating truncated mRNA. This approach has two limitations: (i) it can be technically challenging, and (ii) it only works in vitro, where the concentrations of cellular components differ from concentrations in vivo. We have developed a method to produce stalled ribosomes bearing nascent chains of a specified length by using a 'stall sequence', derived from the Escherichia coli SecM protein, which interacts with residues in the ribosomal exit tunnel to stall SecM translation. When the stall sequence is expressed at the end of nascent chains, stable translation-arrested ribosome complexes accumulate in intact cells or cell-free extracts. SecM-directed stalling is efficient, with negligible effects on viability. This method is straightforward and suitable for producing stalled ribosome complexes in vivo, permitting study of the length-dependent maturation of nascent chains in the cellular milieu.     

Molecular mechanism of drug-dependent ribosome stalling

Inducible expression of the erm erythromycin resistance genes relies on drug-dependent ribosome stalling. The molecular mechanisms underlying stalling are unknown. We used a cell-free translation system to elucidate the contribution of the nascent peptide, the drug, and the ribosome toward formation of the stalled complex during translation of the ermC leader cistron. Toe-printing mapping, selective amino acid labeling, and mutational analyses revealed the peptidyl transferase center (PTC) as the focal point of the stalling mechanism. In the ribosome exit tunnel, the C-terminal sequence of the nascent peptide, critical for stalling, is in the immediate vicinity of the universally conserved A2062 of 23S rRNA. Mutations of this nucleotide eliminate stalling. Because A2062 is located in the tunnel, it may trigger a conformational change in the PTC, responding to the presence of a specific nascent peptide. The cladinose-containing macrolide antibiotic in the tunnel positions the nascent peptide for interaction with the tunnel sensory elements.     

Modulating the activity of the peptidyl transferase center of the ribosome

The peptidyl transferase (PT) center of the ribosome catalyzes two nucleophilic reactions, peptide bond formation between aminoacylated tRNA substrates and, together with release factor, peptide release. Structure and function of the PT center are modulated by binding of aminoacyl-tRNA or release factor, thus providing the basis for the specificity of catalysis. Another way by which the function of the PT center is controlled is signaling from the peptide exit tunnel. The SecM nascent peptide induces ribosome stalling, presumably by inhibition of peptide bond formation. Similarly, the release factor-induced hydrolytic activity of the PT center can be suppressed by the TnaC nascent peptide contained in the exit tunnel. Thus, local and long-range conformational rearrangements can lead to changes in the reaction specificity and catalytic activity of the PT center.     

Prolyl-tRNA(Pro) in the A-site of SecM-arrested ribosomes inhibits the recruitment of transfer-messenger RNA

Translational pausing can lead to cleavage of the A-site codon and facilitate recruitment of the transfer-messenger RNA (tmRNA) (SsrA) quality control system to distressed ribosomes. We asked whether aminoacyl-tRNA binding site (A-site) mRNA cleavage occurs during regulatory translational pausing using the Escherichia coli SecM-mediated ribosome arrest as a model. We find that SecM ribosome arrest does not elicit efficient A-site cleavage, but instead allows degradation of downstream mRNA to the 3'-edge of the arrested ribosome. Characterization of SecM-arrested ribosomes shows the nascent peptide is covalently linked via glycine 165 to tRNA(3Gly) in the peptidyl-tRNA binding site, and prolyl-tRNA(2Pro) is bound to the A-site. Although A-site-cleaved mRNAs were not detected, tmRNA-mediated ssrA tagging after SecM glycine 165 was observed. This tmRNA activity results from sequestration of prolyl-tRNA(2Pro) on overexpressed SecM-arrested ribosomes, which produces a second population of stalled ribosomes with unoccupied A-sites. Indeed, compensatory overexpression of tRNA(2Pro) readily inhibits ssrA tagging after glycine 165, but has no effect on the duration of SecM ribosome arrest. We conclude that, under physiological conditions, the architecture of SecM-arrested ribosomes allows regulated translational pausing without interference from A-site cleavage or tmRNA activities. Moreover, it seems likely that A-site mRNA cleavage is generally avoided or inhibited during regulated ribosome pauses.