Incomplete Polypeptides Research Articles

Peptidyl-tRNA hydrolase is the nascent chain release factor in bacterial ribosome-associated quality control

Rescuing stalled ribosomes often involves their splitting into subunits. In many bacteria, the resultant large subunits bearing peptidyl-tRNAs are processed by the ribosome-associated quality control (RQC) apparatus that extends the C termini of the incomplete nascent polypeptides with polyalanine tails to facilitate their degradation. Although the tailing mechanism is well established, it is unclear how the nascent polypeptides are cleaved off the tRNAs. We show that peptidyl-tRNA hydrolase (Pth), the known role of which has been to hydrolyze ribosome-free peptidyl-tRNA, acts in concert with RQC factors to release nascent polypeptides from large ribosomal subunits. Dislodging from the ribosomal catalytic center is required for peptidyl-tRNA hydrolysis by Pth. Nascent protein folding may prevent peptidyl-tRNA retraction and interfere with the peptide release. However, oligoalanine tailing makes the peptidyl-tRNA ester bond accessible for Pth-catalyzed hydrolysis. Therefore, the oligoalanine tail serves not only as a degron but also as a facilitator of Pth-catalyzed peptidyl-tRNA hydrolysis.

Benefits of co-translational complex assembly for cellular fitness.

Complexes of two or more proteins form many, if not most, of the intracellular "machines" that execute physical and chemical work, and transmit information. Complexes can form from stochastic post-translational interactions of fully formed proteins, but recent attention has shifted to co-translational interactions in which the most common mechanism involves binding of a mature constituent to an incomplete polypeptide emerging from a translating ribosome. Studies in yeast have revealed co-translational interactions during formation of multiple major complexes, and together with recent mammalian cell studies, suggest widespread utilization of the mechanism. These translation-dependent interactions can involve a single or multiple mRNA templates, can be uni- or bi-directional, and can use multi-protein sub-complexes as a binding component. Here, we discuss benefits of co-translational complex assembly including accuracy and efficiency, overcoming hidden interfaces, localized and hierarchical assembly, and reduction of orphan protein degradation, toxicity, and dominant-negative pathogenesis, all serving to improve cell fitness.

Ribosome Rescue Pathways in Bacteria.

Ribosomes that become stalled on truncated or damaged mRNAs during protein synthesis must be rescued for the cell to survive. Bacteria have evolved a diverse array of rescue pathways to remove the stalled ribosomes from the aberrant mRNA and return them to the free pool of actively translating ribosomes. In addition, some of these pathways target the damaged mRNA and the incomplete nascent polypeptide chain for degradation. This review highlights the recent developments in our mechanistic understanding of bacterial ribosomal rescue systems, including drop-off, trans-translation mediated by transfer-messenger RNA and small protein B, ribosome rescue by the alternative rescue factors ArfA and ArfB, as well as Bacillus ribosome rescue factor A, an additional rescue system found in some Gram-positive bacteria, such as Bacillus subtilis. Finally, we discuss the recent findings of ribosome-associated quality control in particular bacterial lineages mediated by RqcH and RqcP. The importance of rescue pathways for bacterial survival suggests they may represent novel targets for the development of new antimicrobial agents against multi-drug resistant pathogenic bacteria.

NEMF mutations that impair ribosome-associated quality control are associated with neuromuscular disease

A hallmark of neurodegeneration is defective protein quality control. The E3 ligase Listerin (LTN1/Ltn1) acts in a specialized protein quality control pathway—Ribosome-associated Quality Control (RQC)—by mediating proteolytic targeting of incomplete polypeptides produced by ribosome stalling, and Ltn1 mutation leads to neurodegeneration in mice. Whether neurodegeneration results from defective RQC and whether defective RQC contributes to human disease have remained unknown. Here we show that three independently-generated mouse models with mutations in a different component of the RQC complex, NEMF/Rqc2, develop progressive motor neuron degeneration. Equivalent mutations in yeast Rqc2 selectively interfere with its ability to modify aberrant translation products with C-terminal tails which assist with RQC-mediated protein degradation, suggesting a pathomechanism. Finally, we identify NEMF mutations expected to interfere with function in patients from seven families presenting juvenile neuromuscular disease. These uncover NEMF’s role in translational homeostasis in the nervous system and implicate RQC dysfunction in causing neurodegeneration.

Aggregation of CAT tails blocks their degradation and causes proteotoxicity in S. cerevisiae.

The Ribosome-associated Quality Control (RQC) pathway co-translationally marks incomplete polypeptides from stalled translation with two signals that trigger their proteasome-mediated degradation. The E3 ligase Ltn1 adds ubiquitin and Rqc2 directs the large ribosomal subunit to append carboxy-terminal alanine and threonine residues (CAT tails). When excessive amounts of incomplete polypeptides evade Ltn1, CAT-tailed proteins accumulate and can self-associate into aggregates. CAT tail aggregation has been hypothesized to either protect cells by sequestering potentially toxic incomplete polypeptides or harm cells by disrupting protein homeostasis. To distinguish between these possibilities, we modulated CAT tail aggregation in Saccharomyces cerevisiae with genetic and chemical tools to analyze CAT tails in aggregated and un-aggregated states. We found that enhancing CAT tail aggregation induces proteotoxic stress and antagonizes degradation of CAT-tailed proteins, while inhibiting aggregation reverses these effects. Our findings suggest that CAT tail aggregation harms RQC-compromised cells and that preventing aggregation can mitigate this toxicity.

CAT-tailing as a fail-safe mechanism for efficient degradation of stalled nascent polypeptides.

Ribosome stalling leads to recruitment of the ribosome quality control complex (RQC), which targets the partially synthesized polypeptide for proteasomal degradation through the action of the ubiquitin ligase Ltn1p. A second core RQC component, Rqc2p, modifies the nascent polypeptide by adding a carboxyl-terminal alanine and threonine (CAT) tail through a noncanonical elongation reaction. Here we examined the role of CAT-tailing in nascent-chain degradation in budding yeast. We found that Ltn1p efficiently accessed only nascent-chain lysines immediately proximal to the ribosome exit tunnel. For substrates without Ltn1p-accessible lysines, CAT-tailing enabled degradation by exposing lysines sequestered in the ribosome exit tunnel. Thus, CAT-tails do not serve as a degron, but rather provide a fail-safe mechanism that expands the range of RQC-degradable substrates.

A tale of CAT tails

Proteostasis When protein translation fails, the incomplete nascent polypeptide is targeted for degradation by the highly conserved ribosome-associated quality control complex (RQC). Mutations in RQC components lead to stress at the cellular level and neurodegeneration at the organismal level. Recent studies have shown that RQC tags partially synthesize proteins with C-terminal alanine and threonine (CAT) tails in an unusual elongation reaction. Working in yeast, Kostova et al. elucidated the role of this process. CAT-tailing is a fail-safe mechanism to ensure the degradation of partially synthesized proteins. The elongation process appears to “push” lysines out of the ribosome exit tunnel, which allows them to be marked by ubiquitin degradation signals. Science , this issue p. [414][1] [1]: /lookup/doi/10.1126/science.aam7787

Comprehensive structural analysis of designed incomplete polypeptide chains of the replicase nonstructural protein 1 from the severe acute respiratory syndrome coronavirus.

The cotranslational folding is recognized as a very cooperative process that occurs after the nearly completion of the polypeptide sequence of a domain. Here we investigated the challenges faced by polypeptide segments of a non-vectorial β-barrel fold. Besides the biological interest behind the SARS coronavirus non-structural protein 1 (nsp1, 117 amino acids), this study model has two structural features that motivated its use in this work: 1- its recombinant production is dependent on the temperature, with greater solubility when expressed at low temperatures. This is an indication of the cotranslational guidance to the native protein conformation. 2- Conversely, nsp1 has a six-stranded, mixed parallel/antiparallel β-barrel with intricate long-range interactions, indicating it will need the full-length protein to fold properly. We used non-denaturing purification conditions that allowed the characterization of polypeptide chains of different lengths, mimicking the landscape of the cotranslational fold of a β-barrel, and avoiding the major technical hindrances of working with the nascent polypeptide bound to the ribosome. Our results showed partially folded states formed as soon as the amino acids of the second β-strand were present (55 amino acids). These partially folded states are different based on the length of polypeptide chain. The native α-helix (amino acids 24–37) was identified as a transient structure (~20–30% propensity). We identified the presence of regular secondary structure after the fourth native β-strand is present (89 amino acids), in parallel to the collapse to a non-native 3D structure. Interestingly the polypeptide sequences of the native strands β2, β3 and β4 have characteristics of α-helices. Our comprehensive analyses support the idea that incomplete polypeptide chains, such as the ones of nascent proteins much earlier than the end of the translation, adopt an abundance of specific transient folds, instead of disordered conformations.

Resolving Nonstop Translation Complexes Is a Matter of Life or Death

Problems during gene expression can result in a ribosome that has translated to the 3' end of an mRNA without terminating at a stop codon, forming a nonstop translation complex. The nonstop translation complex contains a ribosome with the mRNA and peptidyl-tRNA engaged, but because there is no codon in the A site, the ribosome cannot elongate or terminate the nascent chain. Recent work has illuminated the importance of resolving these nonstop complexes in bacteria. Transfer-messenger RNA (tmRNA)-SmpB specifically recognizes and resolves nonstop translation complexes in a reaction known as trans-translation. trans-Translation releases the ribosome and promotes degradation of the incomplete nascent polypeptide and problematic mRNA. tmRNA and SmpB have been found in all bacteria and are essential in some species. However, other bacteria can live without trans-translation because they have one of the alternative release factors, ArfA or ArfB. ArfA recruits RF2 to nonstop translation complexes to promote hydrolysis of the peptidyl-tRNAs. ArfB recognizes nonstop translation complexes in a manner similar to tmRNA-SmpB recognition and directly hydrolyzes the peptidyl-tRNAs to release the stalled ribosomes. Genetic studies indicate that most or all species require at least one mechanism to resolve nonstop translation complexes. Consistent with such a requirement, small molecules that inhibit resolution of nonstop translation complexes have broad-spectrum antibacterial activity. These results suggest that resolving nonstop translation complexes is a matter of life or death for bacteria.

The balance between protein folding and aggregation in the early stages of a protein’s life: role of the ribosome and molecular chaperones (752.6)

Protein biosynthesis takes place within the ribosomal machinery in the presence of the molecular chaperones trigger factor (TF) and DnaK, which are involved in the early stages of a protein’s life in bacteria. To date, the role of the ribosome, DnaK and TF in preventing co‐ and post‐translational protein aggregation is poorly understood. In this study, we used sperm whale apomyoglobin as a model protein to investigate how the ribosome, trigger factor and DnaK affect the balance between protein folding and aggregation, in the context of an E. coli cell free system. We detected both the soluble and insoluble fractions of newly synthesized apomyoglobin in a cell‐free transcription‐translation environment in the presence and absence of TF and DnaK. We took advantage of in situ labeling the N‐terminal methionine via the unnatural BODIPY‐tRNA fMet. Given that the genes encoding TF and DnaK cannot be both knocked out within the same cell strain, we developed a short peptide that can specifically inhibit DnaK activity, and added it into the cell free extract of a tig knock out strain to explore the translation in the absence of both DnaK and trigger factor. We also studied how these two chaperones and the ribosome affect incomplete nascent protein aggregation by examining the solubility of different truncated apomyoglobin constructs. Our results show that elimination of both TF and DnaK activity leads to accumulation of insoluble truncated polypeptides but does not affect the solubility of the full‐length protein. Our findings demonstrate that molecular chaperones are important for preventing any post‐translational aggregation of incomplete polypeptides but the ribosome alone suffices in promoting the successful folding of the full‐length protein.Grant Funding Source: Supported by NSF grant

A second eukaryotic group with mitochondrion-encoded tmRNA

In bacteria, stalled ribosomes are rescued by transfer-mRNA (tmRNA) that catalyzes two steps. First, a non-encoded alanine is added to the incomplete polypeptide chain by the tRNAAla-like portion of tmRNA, and second, the ribosome switches to the mRNA-like domain of tmRNA, thus resuming protein synthesis. Mitochondrial DNA (mtDNA)-encoded mt-tmRNA is so far only known from jakobid protists, but we posit that the corresponding ssrA gene may also reside in other mtDNAs. Here we present a highly sensitive covariance model built from jakobid ssrA genes that identifies previously unrecognized ssrA homologs in mtDNAs of oomycetes. These genes, located in previously unassigned genomic regions, are circular permuted as in α-Protobacteria, implying that pre-tmRNA is processed and the two pieces are held together by non-covalent interactions. RNA-Seq data from Phytophthora sojae confirm predicted processing sites as well as post-transcriptional addition of 3′ CCA, a prerequisite for tmRNAs to be charged with alanine by alanyl-tRNA synthetase. Structure modeling of oomycete tmRNAs infers that the mRNA-like domain is lacking as in jakobids. Features of mitochondrial tmRNAs include the G-U pair at position three of the acceptor stem, a hallmark of bacterial tmRNAs, and a T-loop sequence that differs from that of standard tRNAs and most bacterial tmRNAs, forming alternative, virtually isosteric tertiary interactions with the D-loop. The anticodon stem has two additional G-A base pairs formed between the D-loop and the variable region, shortening the length of the variable region to a single nucleotide.

Novel factor rescues ribosomes trapped on non-stop mRNAs

Ribosomes are trapped at the 3′ ends of mRNAs that lack a natural stop codon. In bacteria, a reaction called trans-translation recycles ribosomes entrapped at such ‘non-stop’ mRNAs. The main player in trans-translation is tmRNA (SsrA-RNA), a bi-functional RNA that acts as both a tRNA and an mRNA. In the trans-translation reaction, alanine-charged tmRNA loads at the ribosomal A-site and translation shifts to the resume codon in tmRNA. Translation of tmRNA stops at a natural stop codon at the end of the small reading frame of tmRNA. In this way, the reaction simultaneously adds a peptide tag to the end of the nascent, incomplete polypeptide and recycles the stalled ribosomes. The peptide tag is recognized by cellular proteases that rapidly degrade the incomplete, potentially harmful polypeptides. The trans-translation reaction is not essential in most bacteria, raising the possibility that ribosomes stalled at non-stop mRNAs can be rescued by alternative routes. In this issue of Molecular Microbiology, Chadani et al. show that a novel translation factor, ArfA, can recycle a ribosome trapped at the 3′ end of a non-stop mRNA in the absence of trans-translation. AfrA is essential in the absence of tmRNA, showing that the two systems work in parallel to resolve stalled ribosomes.

Halting a cellular production line: responses to ribosomal pausing during translation

Cellular protein synthesis is a complex polymerization process carried out by multiple ribosomes translating individual mRNAs. The process must be responsive to rapidly changing conditions in the cell that could cause ribosomal pausing and queuing. In some circumstances, pausing of a bacterial ribosome can trigger translational abandonment via the process of trans-translation, mediated by tmRNA (transfer-messenger RNA) and endonucleases. Together, these factors release the ribosome from the mRNA and target the incomplete polypeptide for destruction. In eukaryotes, ribosomal pausing can initiate an analogous process carried out by the Dom34p and Hbs1p proteins, which trigger endonucleolytic attack of the mRNA, a process termed mRNA no-go decay. However, ribosomal pausing can also be employed for regulatory purposes, and controlled translational delays are used to help co-translational folding of the nascent polypeptide on the ribosome, as well as a tactic to delay translation of a protein while its encoding mRNA is being localized within the cell. However, other responses to pausing trigger ribosomal frameshift events. Recent discoveries are thus revealing a wide variety of mechanisms used to respond to translational pausing and thus regulate the flow of ribosomal traffic on the mRNA population.

Lon Protease Degrades Transfer-Messenger RNA-Tagged Proteins

Bacterial trans translation is activated when translating ribosomes are unable to elongate or terminate properly. Small protein B (SmpB) and transfer-messenger RNA (tmRNA) are the two known factors required for and dedicated to trans translation. tmRNA, encoded by the ssrA gene, is a bifunctional molecule that acts both as a tRNA and as an mRNA during trans translation. The functions of tmRNA ensure that stalled ribosomes are rescued, the causative defective mRNAs are degraded, and the incomplete polypeptides are marked for targeted proteolysis. We present in vivo and in vitro evidence that demonstrates a direct role for the Lon ATP-dependent protease in the degradation of tmRNA-tagged proteins. In an endogenous protein tagging assay, lon mutants accumulated excessive levels of tmRNA-tagged proteins. In a reporter protein tagging assay with lambda-CI-N, the protein product of a nonstop mRNA construct designed to activate trans translation, lon mutant cells efficiently tagged the reporter protein, but the tagged protein exhibited increased stability. Similarly, a green fluorescent protein (GFP) construct containing a hard-coded C-terminal tmRNA tag (GFP-SsrA) exhibited increased stability in lon mutant cells. Most significantly, highly purified Lon preferentially degraded the tmRNA-tagged forms of proteins compared to the untagged forms. Based on these results, we conclude that Lon protease participates directly in the degradation of tmRNA-tagged proteins.

Codon-specific and general inhibition of protein synthesis by the tRNA-sequestering minigenes

The expression of minigenes in bacteria inhibits protein synthesis and cell growth. Presumably, the translating ribosomes, harboring the peptides as peptidyl-tRNAs, pause at the last sense codon of the minigene directed mRNAs. Eventually, the peptidyl-tRNAs drop off and, under limiting activity of peptidyl-tRNA hydrolase, accumulate in the cells reducing the concentration of specific aminoacylable tRNA. Therefore, the extent of inhibition is associated with the rate of starvation for a specific tRNA. Here, we used minigenes harboring various last sense codons that sequester specific tRNAs with different efficiency, to inhibit the translation of reporter genes containing, or not, these codons. A prompt inhibition of the protein synthesis directed by genes containing the codons starved for their cognate tRNA (hungry codons) was observed. However, a non-specific in vitro inhibition of protein synthesis, irrespective of the codon composition of the gene, was also evident. The degree of inhibition correlated directly with the number of hungry codons in the gene. Furthermore, a tRNA Arg4-sequestering minigene promoted the production of an incomplete β-galactosidase polypeptide interrupted, during bacterial polypeptide chain elongation at sites where AGA codons were inserted in the lacZ gene suggesting ribosome pausing at the hungry codons.

Effect of Hsp70 Chaperone on the Folding and Misfolding of Polypeptides Modeling an Elongating Protein Chain

Virtually nothing is known about the interaction of co-translationally active chaperones with nascent polypeptides and the resulting effects on peptide conformation and folding. We have explored this issue by NMR analysis of apomyoglobin N-terminal fragments of increasing length, taken as models for different stages of protein biosynthesis, in the absence and presence of the substrate binding domain of Escherichia coli Hsp70, DnaK-beta. The incomplete polypeptides misfold and self-associate under refolding conditions. In the presence of DnaK-beta, however, formation of the original self-associated species is completely or partially prevented. Chaperone interaction with incomplete protein chains promotes a globally unfolded dynamic DnaK-beta-bound state, which becomes folding-competent only upon incorporation of the residues corresponding to the C-terminal H helix. The chaperone does not bind the full-length protein at equilibrium. However, its presence strongly disfavors the kinetic accessibility of misfolding side-routes available to the full-length chain. This work supports the role of DnaK as a "holder" for incomplete N-terminal polypeptides. However, as the chain approaches its full-length status, the tendency to intramolecularly bury non-polar surface efficiently outcompetes chaperone binding. Under these conditions, DnaK serves as a "folding enhancer" by supporting folding of a population of otherwise folding-incompetent full-length protein chains.

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Ribosome-associated quality-control (RQC) surveys incomplete nascent polypeptides produced by interrupted translation. Central players in RQC are the human ribosome- and tRNA-binding protein, NEMF, and its orthologs, yeast Rqc2 and bacterial RqcH, which sense large ribosomal subunits obstructed with nascent chains and then promote nascent-chain proteolysis. In canonical eukaryotic RQC, NEMF stabilizes the LTN1/Listerin E3 ligase binding to obstructed ribosomal subunits for nascent-chain ubiquitylation. Furthermore, NEMF orthologs across evolution modify nascent chains by mediating C-terminal, untemplated polypeptide elongation. In eukaryotes, this process exposes ribosome-buried nascent-chain lysines, the ubiquitin acceptor sites, to LTN1. Remarkably, in both bacteria and eukaryotes, C-terminal tails also have an extra-ribosomal function as degrons. Here, we discuss recent findings on RQC mechanisms and briefly review how ribosomal stalling is sensed upstream of RQC, including via ribosome collisions, from an evolutionary perspective. Because RQC defects impair cellular fitness and cause neurodegeneration, this knowledge provides a framework for pathway-related biology and disease studies.

Proteolysis in prokaryotes: protein quality control and regulatory principles.

Proteolysis in prokaryotes: protein quality control and regulatory principles.

SsrA-mediated trans-translation plays a role in mRNA quality control by facilitating degradation of truncated mRNAs

An important unsolved question regarding the bacterial SsrA system is the fate of target mRNAs replaced by SsrA RNA during trans-translation. The aim of the present study is to address the potential role of SsrA system in mRNA quality control, focusing on truncated mRNAs that are expected to arise from 3'-to-5' exonucleolytic attack. We found that significant amounts of truncated mRNAs and polypeptides were produced from genes lacking a rho-independent terminator in SsrA-deficient cells. These truncated mRNAs, hence truncated polypeptides, were no longer observed in the presence of SsrA RNA. The data indicate that the SsrA system facilitates degradation of "nonstop" mRNAs by presumably removing the stalled ribosomes. Furthermore, analysis of affinity-purified proteins indicated that truncated polypeptides could be produced even from a gene with an intact rho-independent terminator, although less efficiently, implying that C-terminally truncated proteins and 3'-truncated mRNA may be produced from virtually all protein-coding genes. We conclude that the SsrA system not only promotes the degradation of incomplete polypeptides but also minimizes the synthesis of incomplete polypeptides by facilitating the degradation of truncated mRNAs that are produced in cells.

Three genes showing distinct regulatory patterns encode the asparagine synthetase of sunflower (Helianthus annuus).

• Asparagine metabolism in sunflower (Helianthus annuus) was investigated by cDNA cloning, sequence characterization and expression analysis of three genes encoding different isoforms of asparagine synthetase (AS, EC 6.3.5.4). • The AS-coding sequences were searched for in leaves, roots and cotyledons by using a methodology based on the simultaneous amplification of different cDNAs. Three distinct AS-coding genes, HAS1, HAS1.1 and HAS2, were identified. • HAS1 and HAS1.1 are twin genes with closely related sequences that share some regulatory features. By contrast, HAS2 is a singular sequence that encodes an incomplete AS polypeptide and shows an unusual regulation. The functionality of both the complete HAS1 and the truncated HAS2 proteins was demonstrated by complementation assays. Northern analysis revealed that HAS1, HAS1.1 and HAS2 were differentially regulated dependent on the organ, the physiological status, the developmental stage and the light conditions. • Asparagine synthetase from sunflower is encoded by a small gene family whose members have achieved a significant degree of specialization to cope with the major situations requiring asparagine synthesis.