Arrest Peptide Research Articles

Effect of Translation-Enhancing Nascent SKIK Peptide on the Arrest Peptides Containing Consecutive Proline.

Ribosome arrest peptides (RAPs) such as the SecM arrest peptide (SecM AP: FSTPVWISQAQGIRAGP) and WPPP with consecutive Pro residues are known to induce translational stalling in Escherichia coli. We demonstrate that the translation-enhancing SKIK peptide tag, which consists of four amino acid residues Ser-Lys-Ile-Lys, effectively alleviates translational arrest caused by WPPP. Moreover, the proximity between SKIK and WPPP significantly influences the extent of this alleviation, observed in both PURE cell-free protein synthesis and in vivo protein production systems, resulting in a substantial increase in the yield of proteins containing such RAPs. Furthermore, we unveil that nascent SKIK peptide tag and translation elongation factor P (EF-P) alleviate ribosome stalling in consecutive-Pro-rich protein to synergistically promote translation. A kinetic analysis based on the generation of superfolder green fluorescent protein under in vitro translation reaction reveals that the ribosome turnover is enhanced by more than 10-fold when the SKIK peptide tag is positioned immediately upstream of the SecM AP sequence. Our findings provide valuable insights into optimizing protein production processes, which are essential for advancing synthetic biology applications.

Single-residue effects on the behavior of a nascent polypeptide chain inside the ribosome exit tunnel.

Nascent polypeptide chains (NCs) are extruded from the ribosome through an exit tunnel (ET) traversing the large ribosomal subunit. The ET's irregular and chemically complex wall allows for various NC-ET interactions. Translational arrest peptides (APs) bind in the ET to induce translational arrest, a property that can be exploited to study NC-ET interactions by Force Profile Analysis (FPA). We employed FPA and molecular dynamics (MD) simulations to investigate how individual residues placed in a glycine-serine repeat segment within an AP-stalled NC interact with the ET to exert a pulling force on the AP and release stalling. Our results indicate that large and hydrophobic residues generate a pulling force on the NC when placed ≳10 residues away from the peptidyl transfer center (PTC). Moreover, an asparagine placed 12 residues from the PTC makes a specific stabilizing interaction with the tip of ribosomal protein uL22 that reduces the pulling force on the NC, while a lysine or leucine residue in the same position increases the pulling force. Finally, the MD simulations suggest how the Mannheimia succiniproducens SecM AP interacts with the ET to promote translational stalling.

Patchy and widespread distribution of bacterial translation arrest peptides associated with the protein localization machinery

Regulatory arrest peptides interact with specific residues on bacterial ribosomes and arrest their own translation. Here, we analyse over 30,000 bacterial genome sequences to identify additional Sec/YidC-related arrest peptides, followed by in vivo and in vitro analyses. We find that Sec/YidC-related arrest peptides show patchy, but widespread, phylogenetic distribution throughout the bacterial domain. Several of the identified peptides contain distinct conserved sequences near the C-termini, but are still able to efficiently stall bacterial ribosomes in vitro and in vivo. In addition, we identify many arrest peptides that share an R-A-P-P-like sequence, suggesting that this sequence might serve as a common evolutionary seed to overcome ribosomal structural differences across species.

The SecM arrest peptide traps a pre-peptide bond formation state of the ribosome

Nascent polypeptide chains can induce translational stalling to regulate gene expression. This is exemplified by the E. coli secretion monitor (SecM) arrest peptide that induces translational stalling to regulate expression of the downstream encoded SecA, an ATPase that co-operates with the SecYEG translocon to facilitate insertion of proteins into or through the cytoplasmic membrane. Here we present the structure of a ribosome stalled during translation of the full-length E. coli SecM arrest peptide at 2.0 Å resolution. The structure reveals that SecM arrests translation by stabilizing the Pro-tRNA in the A-site, but in a manner that prevents peptide bond formation with the SecM-peptidyl-tRNA in the P-site. By employing molecular dynamic simulations, we also provide insight into how a pulling force on the SecM nascent chain can relieve the SecM-mediated translation arrest. Collectively, the mechanisms determined here for SecM arrest and relief are also likely to be applicable for a variety of other arrest peptides that regulate components of the protein localization machinery identified across a wide range of bacteria lineages.

RAPP-containing arrest peptides induce translational stalling by short circuiting the ribosomal peptidyltransferase activity

Arrest peptides containing RAPP (ArgAlaProPro) motifs have been discovered in both Gram-positive and Gram-negative bacteria, where they are thought to regulate expression of important protein localization machinery components. Here we determine cryo-EM structures of ribosomes stalled on RAPP arrest motifs in both Bacillus subtilis and Escherichia coli. Together with molecular dynamics simulations, our structures reveal that the RAPP motifs allow full accommodation of the A-site tRNA, but prevent the subsequent peptide bond from forming. Our data support a model where the RAP in the P-site interacts and stabilizes a single hydrogen atom on the Pro-tRNA in the A-site, thereby preventing an optimal geometry for the nucleophilic attack required for peptide bond formation to occur. This mechanism to short circuit the ribosomal peptidyltransferase activity is likely to operate for the majority of other RAPP-like arrest peptides found across diverse bacterial phylogenies.

RPL26/uL24 UFMylation is essential for ribosome-associated quality control at the endoplasmic reticulum

Ribosomes that stall while translating cytosolic proteins are incapacitated by incomplete nascent chains, termed "arrest peptides" (APs) that are destroyed by the ubiquitin proteasome system (UPS) via a process known as the ribosome-associated quality control (RQC) pathway. By contrast, APs on ribosomes that stall while translocating secretory proteins into the endoplasmic reticulum (ER-APs) are shielded from cytosol by the ER membrane and the tightly sealed ribosome-translocon junction (RTJ). How this junction is breached to enable access of cytosolic UPS machinery and 26S proteasomes to translocon- and ribosome-obstructing ER-APs is not known. Here, we show that UPS and RQC-dependent degradation of ER-APs strictly requires conjugation of the ubiquitin-like (Ubl) protein UFM1 to 60S ribosomal subunits at the RTJ. Therefore, UFMylation of translocon-bound 60S subunits modulates the RTJ to promote access of proteasomes and RQC machinery to ER-APs.

Nascent MSKIK peptide cancels ribosomal stalling by arrest peptides in Escherichia coli

The insertion of the DNA sequence encoding SKIK peptide adjacent to the M start codon of a difficult-to-express protein enhances protein production in Escherichia coli. In this report, we reveal that the increased production of the SKIK-tagged protein is not due to codon usage of the SKIK sequence. Furthermore, we found that insertion of SKIK or MSKIK just before the SecM arrest peptide (FSTPVWISQAQGIRAGP), which causes ribosomal stalling on mRNA, greatly increased the production of the protein containing the SecM arrest peptide in the E.coli-reconstituted cell-free protein synthesis system (PURE system). A similar translation enhancement phenomenon by MSKIK was observed for the CmlA leader peptide, a ribosome arrest peptide, whose arrest is induced by chloramphenicol. These results strongly suggest that the nascent MSKIK peptide prevents or releases ribosomal stalling immediately following its generation during the translation process, resulting in an increase of protein production.

Cotranslational folding and assembly of the dimeric Escherichia coli inner membrane protein EmrE

In recent years, it has become clear that many homo- and heterodimeric cytoplasmic proteins in both prokaryotic and eukaryotic cells start to dimerize cotranslationally (i.e., while at least one of the two chains is still attached to the ribosome). Whether this is also possible for integral membrane proteins is, however, unknown. Here, we apply force profile analysis (FPA)-a method where a translational arrest peptide (AP) engineered into the polypeptide chain is used to detect force generated on the nascent chain during membrane insertion-to demonstrate cotranslational interactions between a fully membrane-inserted monomer and a nascent, ribosome-tethered monomer of the Escherichia coli inner membrane protein EmrE. Similar cotranslational interactions are also seen when the two monomers are fused into a single polypeptide. Further, we uncover an apparent intrachain interaction between E14 in transmembrane helix 1 (TMH1) and S64 in TMH3 that forms at a precise nascent chain length during cotranslational membrane insertion of an EmrE monomer. Like soluble proteins, inner membrane proteins thus appear to be able to both start to fold and start to dimerize during the cotranslational membrane insertion process.

Proteomic analysis of nascent polypeptide chains that potentially induce translational pausing during elongation.

Currently, proteins equipped with "ribosomal arrest peptides" (RAPs) that regulate the expression of downstream genes and their own activity by pausing their own translation during elongation are extensively studied. However, studies focusing on RAP have been conducted primarily in prokaryotic cells; studies on eukaryotic cells, especially mammalian cells, are limited. In the present study, we comprehensively examined translationally arrested nascent polypeptides to gain novel insights into RAPs in mammalian cells. Cetyltrimethylammonium bromide was used to obtain nascent polypeptide chains that were translationally arrested during translation elongation. After proteomic analysis, additional screening by discriminating according to amino acid residues at the C-terminal end revealed several novel RAP candidates. Our method can be applied for comprehensive RAP studies in mammalian cells.

AP Profiling: Resolving Co‐Translational Protein Folding Pathways and Chaperone Interactions In Vivo

Co‐translational folding and molecular chaperone action are crucial for productive protein folding in the cell, but how these processes shape folding pathways remains largely unknown. We have utilized translation arrest peptides (APs) to monitor co‐translational folding in live bacterial cells, combined with single‐molecule optical tweezers experiments, to define the co‐translational folding pathway of the GTPase domain (G‐domain) from E. coli elongation factor G (EF‐G). Surprisingly, the 293 amino acid long domain remains unfolded, without forming stable intermediate structures, until it is fully extruded from the ribosome. The full‐length G‐domain transitions to its stable native structure via obligate folding intermediates both in isolation and while bound to the ribosome. Folding therefore follows a strictly sequential pathway that initiates at the very C‐terminus, which is likely imposed by the structure and topology of the G‐domain from EF‐G. Consequently, folding and synthesis proceed in opposite directions. G‐domains represent a common element in a number of multi‐domain proteins. To determine whether their folding pathways are conserved, we have combined our AP approach with cell sorting and deep sequencing into a method that we term “AP profiling”. Homologous G‐domains exhibited distinct folding patterns that are conserved across distant bacterial species. Individual deletion of the primary nascent chain‐binding chaperones, trigger factor and DnaK (Hsp70), resulted in numerous localized changes to co‐translational folding while preserving overall G‐domain folding, highlighting the functional redundancy of cellular chaperone systems. In summary, we have developed AP profiling as a technique for monitoring co‐translational folding in the native cellular environment with high throughput. Combined with single‐molecule force spectroscopy, AP profiling yields a unique view of co‐translational folding and chaperone function.

Probing Interplays between Human XBP1u Translational Arrest Peptide and 80S Ribosome.

The ribosome stalling mechanism is a crucial biological process, yet its atomistic underpinning is still elusive. In this framework, the human XBP1u translational arrest peptide (AP) plays a central role in regulating the unfolded protein response (UPR) in eukaryotic cells. Here, we report multimicrosecond all-atom molecular dynamics simulations designed to probe the interactions between the XBP1u AP and the mammalian ribosome exit tunnel, both for the wild type AP and for four mutant variants of different arrest potencies. Enhanced sampling simulations allow investigating the AP release process of the different variants, shedding light on this complex mechanism. The present outcomes are in qualitative/quantitative agreement with available experimental data. In conclusion, we provide an unprecedented atomistic picture of this biological process and clear-cut insights into the key AP–ribosome interactions.

Structural basis of l-tryptophan-dependent inhibition of release factor 2 by the TnaC arrest peptide.

In Escherichia coli, elevated levels of free l-tryptophan (l-Trp) promote translational arrest of the TnaC peptide by inhibiting its termination. However, the mechanism by which translation-termination by the UGA-specific decoding release factor 2 (RF2) is inhibited at the UGA stop codon of stalled TnaC-ribosome-nascent chain complexes has so far been ambiguous. This study presents cryo-EM structures for ribosomes stalled by TnaC in the absence and presence of RF2 at average resolutions of 2.9 and 3.5 Å, respectively. Stalled TnaC assumes a distinct conformation composed of two small α-helices that act together with residues in the peptide exit tunnel (PET) to coordinate a single L-Trp molecule. In addition, while the peptidyl-transferase center (PTC) is locked in a conformation that allows RF2 to adopt its canonical position in the ribosome, it prevents the conserved and catalytically essential GGQ motif of RF2 from adopting its active conformation in the PTC. This explains how translation of the TnaC peptide effectively allows the ribosome to function as a L-Trp-specific small-molecule sensor that regulates the tnaCAB operon.

Cotranslational Translocation and Folding of a Periplasmic Protein Domain in Escherichia coli

In Gram-negative bacteria, periplasmic domains in inner membrane proteins are cotranslationally translocated across the inner membrane through the SecYEG translocon. To what degree such domains also start to fold cotranslationally is generally difficult to determine using currently available methods. Here, we apply Force Profile Analysis (FPA) – a method where a translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide – to follow the cotranslational translocation and folding of the large periplasmic domain of the E. coli inner membrane protease LepB in vivo. Membrane insertion of LepB’s two N-terminal transmembrane helices is initiated when their respective N-terminal ends reach 45–50 residues away from the peptidyl transferase center (PTC) in the ribosome. The main folding transition in the periplasmic domain involves all but the ~15 most C-terminal residues of the protein and happens when the C-terminal end of the folded part is ~70 residues away from the PTC; a smaller putative folding intermediate is also detected. This implies that wildtype LepB folds post-translationally in vivo, and shows that FPA can be used to study both co- and post-translational protein folding in the periplasm.

Force transduction creates long-ranged coupling in ribosomes stalled by arrest peptides

Force-sensitive arrest peptides regulate protein biosynthesis by stalling the ribosome as they are translated. Synthesis can be resumed when the nascent arrest peptide experiences a pulling force of sufficient magnitude to break the stall. Efficient stalling is dependent on the specific identity of a large number of amino acids, including amino acids that are tens of angstroms away from the peptidyl transferase center (PTC). The mechanism of force-induced restart and the role of these essential amino acids far from the PTC is currently unknown. We use hundreds of independent molecular dynamics trajectories spanning over 120 μs in combination with kinetic analysis to characterize multiple barriers along the force-induced restart pathway for the arrest peptide SecM. We find that the essential amino acids far from the PTC play a major role in controlling the transduction of applied force. In successive states along the stall-breaking pathway, the applied force propagates up the nascent chain until it reaches the C-terminus of SecM and the PTC, inducing conformational changes that allow for restart of translation. A similar mechanism of force propagation through multiple states is observed in the VemP stall-breaking pathway, but secondary structure in VemP allows for heterogeneity in the order of transitions through intermediate states. Results from both arrest peptides explain how residues that are tens of angstroms away from the catalytic center of the ribosome impact stalling efficiency by mediating the response to an applied force and shielding the amino acids responsible for maintaining the stalled state of the PTC.

Search for translation arrest peptides encoded upstream of genes for components of protein localization pathways.

Regulatory nascent peptides participate in the regulation of cellular functions by the mechanisms involving regulated translation arrest. A class of them in bacteria, called monitoring substrates, feedback-regulates the expression of a specific component of protein localization machinery. Three monitoring substrates, SecM, MifM and VemP have previously been identified. Here, we attempt at identifying additional arrest peptides in bacteria. Our bioinformatic searches over more than 400 bacterial genomic sequences for proteins that have the common characteristic features shared by the known monitoring substrates and subsequent in vitro and in vivo characterization of the highlighted sequences allowed the identification of three arrest peptides termed ApcA, ApdA and ApdP. ApcA and ApdA homologs are conserved among a subset of actinobacteria, whereas ApdP has homologs in a subset of α-proteobacteria. We demonstrate that these arrest peptides, in their ribosome-tethered nascent states, inhibit peptidyl transfer. The elongation arrest occurs at a specific codon near the 3′ end of the coding region, in a manner depending on the amino acid sequence of the nascent chain. Interestingly, the arrest sequences of ApcA, ApdA and ApdP share a sequence R-A-P-G/P that is essential for the elongation arrest.

Translocon-Associated Protein Complex (TRAP) is Crucial for Co-Translational Translocation of Pre-Proinsulin

Many unanswered questions remain in understanding the biosynthesis of the peptide hormone insulin. Here we elucidate new aspects in the mechanism of co-translational translocation initiation of pre-proinsulin in the endoplasmic reticulum. We utilize a translational arrest peptide derived from the x-box-binding protein (Xbp1) to induce ribosomal stalling and generate translocation intermediates. We find that the insulin signal sequence is rather weakly gating and requires the assistance of auxiliary translocon components to initiate translocation. Probing the translational intermediates with chemical crosslinking, we identified an early interaction with the translocon-associated protein (TRAP) complex. The TRAPβ subunit interacts with pre-proinsulin before the peptide enters the Sec61 translocon channel in a signal sequence-dependent manner. We describe the substrate sequence determinants that are recognized by TRAP on the cytosolic site of the membrane to facilitate substrate-specific opening of the Sec61 translocon channel. Our findings support the hypothesis that the TRAP-dependence is in part determined by the content of glycine and proline residues mainly within the signal sequence.

Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli.

Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding‐induced forces acting on the nascent polypeptide, have so far been limited mainly to small domains of cytosolic proteins that fold in close proximity to the translating ribosome. In this study, we investigate the cotranslational folding of the periplasmic, disulfide bond‐containing Escherichia coli protein alkaline phosphatase (PhoA) in a wild‐type strain background and a strain background devoid of the periplasmic thiol: disulfide interchange protein DsbA. We find that folding‐induced forces can be transmitted via the nascent chain from the periplasm to the polypeptide transferase center in the ribosome, a distance of ~160 Å, and that PhoA appears to fold cotranslationally via at least two disulfide‐stabilized folding intermediates. Thus, Force Profile Analysis can be used to study cotranslational folding of proteins in an extra‐cytosolic compartment, like the periplasm.

The folding and unfolding behavior of ribonuclease H on the ribosome

The health of a cell depends on accurate translation and proper protein folding, whereas misfolding can lead to aggregation and disease. The first opportunity for a protein to fold occurs during translation, when the ribosome and surrounding environment can affect the nascent chain energy landscape. However, quantifying these environmental effects is challenging because ribosomal proteins and rRNA preclude most spectroscopic measurements of protein energetics. Here, we have applied two gel-based approaches, pulse proteolysis and force-profile analysis, to probe the folding and unfolding pathways of RNase H (RNH) nascent chains stalled on the prokaryotic ribosome in vitro. We found that ribosome-stalled RNH has an increased unfolding rate compared with free RNH. Because protein stability is related to the ratio of the unfolding and folding rates, this increase completely accounts for the observed change in protein stability and indicates that the folding rate is unchanged. Using arrest peptide–based force-profile analysis, we assayed the force generated during the folding of RNH on the ribosome. Surprisingly, we found that population of the RNH folding intermediate is required to generate sufficient force to release a stall induced by the SecM stalling sequence and that readthrough of SecM directly correlates with the stability of the RNH folding intermediate. Together, these results imply that the folding pathway of RNH is unchanged on the ribosome. Furthermore, our findings indicate that the ribosome promotes RNH unfolding while the nascent chain is proximal to the ribosome, which may limit the deleterious effects of RNH misfolding and assist in folding fidelity.

Supporting data on prion protein translocation mechanism revealed by pulling force studies.

The Prion protein (PrP) is a highly conserved cell surface glycoprotein. To enter the secretory pathway, the PrP precursor relies on the Sec61 complex and multiple accessory factors all gathering at the membrane of the Endoplasmic reticulum (ER). PrP topogenesis results in the formation of different PrP isoforms. Aside from the typical secretory variant (SecPrP) different pathognomonic, membrane-embedded variants (NtmPrP and CtmPrP) that are associated with neurodegenerative diseases can be found [1]. In this article, we provide supportive data related to “Prion Protein Translocation Mechanism Revealed by Pulling Force Studies” (Kriegler et al., May 2020)[2], where we utilize Xbp1 arrest peptide (AP)-mediated ribosomal stalling to study the co-translational folding experienced by PrP during its insertion into the ER. We measure translocation efficiency and characterize the force exerted on PrP nascent chain so called “pulling force profile”. Here, we describe the method of AP-mediated ribosomal stalling assay together with additional experimental data to the main article. Furthermore, we describe the combination of AP-mediated ribosomal stalling and semi-permeabilized Hela cells (SPCs) as ER membrane source. Using this experimental set-up one can directly determine the contribution of a specific membrane component, e.g. subunits of the ER protein translocase, as pulling factor exerting force on the PrP nascent chain.The data presented here covers (a) the SDS-PAGE gel images visualized by autoradiography, (b) quantification of the different populations of PrP species observed in the AP-mediated ribosomal stalling method, and (c) calculation formulas of the pulling force profiles measured in SPCs in comparison to dog pancreas microsomes as ER membrane donor. Finally, Western Blot analysis and quantification of siRNA knockdown levels compared to control conditions of various translocation components are shown.

Prion Protein Translocation Mechanism Revealed by Pulling Force Studies

The mammalian prion protein (PrP) engages with the ribosome–Sec61 translocation channel complex to generate different topological variants that are either physiological, or involved in neurodegenerative diseases. Here, we describe cotranslational folding and translocation mechanisms of PrP coupled to an Xbp1-based arrest peptide as folding sensor, to measure forces acting on PrP nascent chain. Our data reveal two main pulling events followed by a minor third one exerted on the nascent chains during their translocation.Using those force landscapes, we show that a specific sequence within an intrinsically disordered region, containing a polybasic and glycine-proline rich residues, modulates the second pulling event by interacting with TRAP complex. This work also delineates the sequence of events involved in generation of PrP toxic transmembrane topologies during its synthesis. Our results shed new insight into the folding of such a topological complex protein, where marginal pulling by the signal sequence, together with the flanking downstream sequence in the mature domain, primarily drives an overall inefficient translocation resulting in the nascent chain to adopt alternative topologies.