Protein Synthesis Quality Control Research Articles

Mitochondrial protein synthesis quality control.

Human mitochondrial DNA is one of the most simplified cellular genomes and facilitates compartmentalized gene expression. Within the organelle, there is no physical barrier to separate transcription and translation, nor is there evidence that quality control surveillance pathways are active to prevent translation on faulty mRNA transcripts. Mitochondrial ribosomes synthesize 13 hydrophobic proteins that require co-translational insertion into the inner membrane of the organelle. To maintain the integrity of the inner membrane, which is essential for organelle function, requires responsive quality control mechanisms to recognize aberrations in protein synthesis. In this review, we explore how defects in mitochondrial protein synthesis can arise due to the culmination of inherent mistakes that occur throughout the steps of gene expression. In turn, we examine the stepwise series of quality control processes that are needed to eliminate any mistakes that would perturb organelle homeostasis. We aim to provide an integrated view on the quality control mechanisms of mitochondrial protein synthesis and to identify promising avenues for future research.

Quality control of protein synthesis in the early elongation stage

In the early stage of bacterial translation, peptidyl-tRNAs frequently dissociate from the ribosome (pep-tRNA drop-off) and are recycled by peptidyl-tRNA hydrolase. Here, we establish a highly sensitive method for profiling of pep-tRNAs using mass spectrometry, and successfully detect a large number of nascent peptides from pep-tRNAs accumulated in Escherichia coli pthts strain. Based on molecular mass analysis, we found about 20% of the peptides bear single amino-acid substitutions of the N-terminal sequences of E. coli ORFs. Detailed analysis of individual pep-tRNAs and reporter assay revealed that most of the substitutions take place at the C-terminal drop-off site and that the miscoded pep-tRNAs rarely participate in the next round of elongation but dissociate from the ribosome. These findings suggest that pep-tRNA drop-off is an active mechanism by which the ribosome rejects miscoded pep-tRNAs in the early elongation, thereby contributing to quality control of protein synthesis after peptide bond formation.

Comparative Analysis of the Global Transcriptomic Response to Oxidative Stress of Bacillus anthracis htrA-Disrupted and Parental Wild Type Strains.

We previously demonstrated that the HtrA (High Temperature Requirement A) protease/chaperone active in the quality control of protein synthesis, represents an important virulence determinant of Bacillus anthracis. Virulence attenuation of htrA-disrupted Bacillus anthracis strains was attributed to susceptibility of ΔhtrA strains to stress insults, as evidenced by affected growth under various stress conditions. Here, we report a comparative RNA-seq transcriptomic study generating a database of differentially expressed genes in the B. anthracis htrA-disrupted and wild type parental strains under oxidative stress. The study demonstrates that, apart from protease and chaperone activities, HtrA exerts a regulatory role influencing expression of more than 1000 genes under stress. Functional analysis of groups or individual genes exhibiting strain-specific modulation, evidenced (i) massive downregulation in the ΔhtrA and upregulation in the WT strains of various transcriptional regulators, (ii) downregulation of translation processes in the WT strain, and (iii) downregulation of metal ion binding functions and upregulation of sporulation-associated functions in the ΔhtrA strain. These modulated functions are extensively discussed. Fifteen genes uniquely upregulated in the wild type strain were further interrogated for their modulation in response to other stress regimens. Overexpression of one of these genes, encoding for MazG (a nucleoside triphosphate pyrophosphohydrolase involved in various stress responses in other bacteria), in the ΔhtrA strain resulted in partial alleviation of the H2O2-sensitive phenotype.

SmpB and tmRNA Orchestrate Purine Pathway for the Trimethoprim Resistance in Aeromonas veronii

Small protein B(SmpB) cooperates with transfer-messenger RNA (tmRNA) for trans-translation to ensure the quality control of protein synthesis in prokaryotes. Furthermore, they regulate cell metabolism separately. According to research, SmpB functions as a transcription factor, and tmRNA acts as a small RNA. Purine pathway has been reported to be related to trimethoprim resistance, including hypoxanthine synthesis, adenosine metabolism and guanosine metabolism. Another reason of drug tolerance is the efflux pump of the bacterium. In transcriptomic data, it was shown that the expression of some related enzymes in adenosine metabolism were raised significantly in smpB deletion strain than that of wild type, which led to the differential trimethoprim resistance of Aeromonas veronii (A. veronii). Furthermore, the metabolic products of adenosine AMP, cAMP, and deoxyadenosine were accumulated significantly. However, the expressions of the enzymes related to hypoxanthine synthesis and guanosine metabolism were elevated significantly in ssrA (small stable RNA, tmRNA) deletion strain, which eventually caused an augmented metabolic product xanthine. In addition, the deletion of ssrA also affected the significant downregulations of efflux pump acrA/acrB. The minimal inhibitory concentrations (MIC) were overall decreased after the trimethoprim treatment to the wild type, ΔsmpB and ΔssrA. And the difference in sensitivity between ΔsmpB and ΔssrA was evident. The MIC of ΔsmpB was descended significantly than those of wild type and ΔssrA in M9 medium supplemented with 1 mM adenosine, illustrating that the adenosine metabolism pathway was principally influenced by SmpB. Likewise, the strain ΔssrA conferred more sensitivity than wild type and ΔsmpB in M9 medium supplemented with 1mM guanosine. By overexpressing acrA/acrB, the tolerance to trimethoprim was partially recovered in ΔssrA. These results revealed that SmpB and tmRNA acted on different branches in purine metabolism, conferring the diverse trimethoprim resistance to A. veronii. This study suggests that the trans-translation system might be an effective target in clinical treatment of A. veronii and other multi-antibiotic resistance bacteria with trimethoprim.

Solution structure and backbone dynamics for S1 domain of ribosomal protein S1 from Mycobacterium tuberculosis.

The pro-drug pyrazinamide is hydrolyzed to pyrazinoic acid (POA) in its use for the treatment of tuberculosis. As a molecule with bactericidal activity, POA binds to the C-terminal S1 domain of ribosomal protein S1 from Mycobacterium tuberculosis (MtRpsACTD_S1) to inhibit trans-translation. Trans-translation is a critical component of protein synthesis quality control, and is mediated by transfer-messenger RNA. Here, we have determined the solution structure of MtRpsACTD_S1(280-368), and analyzed its structural dynamics by NMR spectroscopy. The solution structure of MtRpsACTD_S1(280-368) mainly consists of five anti-parallel β strands, two α helices, and two 310 helices. Backbone dynamics reveals that the overall structure of MtRpsACTD_S1(280-368) is rigid, but segment L326-V333 undergoes large amplitude fluctuations on picosecond to nanosecond time scales. In addition, residues V321, H322, V331 and D335 with large Rex values exhibit significant chemical or conformational exchange on microsecond to millisecond time scale. Titration of the truncated MtRpsACTD_S1(280-368) with POA shows similar characteristics to titration of MtRpsACTD_S1(280-438) with POA. In addition, diverse length fragments of MtRpsACTD_S1 show various HN resonance signals, and we find that the interaction of MtRpsA(369-481) with MtRpsACTD_S1(280-368) [Kd = (4.25 ± 0.15) mM] is responsible for the structural difference between MtRpsACTD_S1(280-368) and MtRpsACTD_S1. This work may shed light on the underlying molecular mechanism of MtRpsACTD recognizing and binding POA or mRNA, as well as the detailed mechanism of interactions between MtRpsACTD_S1(280-368) and the additional C-terminal MtRpsA(369-481).

Mitochondrial stress response triggered by defects in protein synthesis quality control.

Mitochondria have a compartmentalized gene expression system dedicated to the synthesis of membrane proteins essential for oxidative phosphorylation. Responsive quality control mechanisms are needed to ensure that aberrant protein synthesis does not disrupt mitochondrial function. Pathogenic mutations that impede the function of the mitochondrial matrix quality control protease complex composed of AFG3L2 and paraplegin cause a multifaceted clinical syndrome. At the cell and molecular level, defects to this quality control complex are defined by impairment to mitochondrial form and function. Here, we establish the etiology of these phenotypes. We show how disruptions to the quality control of mitochondrial protein synthesis trigger a sequential stress response characterized first by OMA1 activation followed by loss of mitochondrial ribosomes and by remodelling of mitochondrial inner membrane ultrastructure. Inhibiting mitochondrial protein synthesis with chloramphenicol completely blocks this stress response. Together, our data establish a mechanism linking major cell biological phenotypes of AFG3L2 pathogenesis and show how modulation of mitochondrial protein synthesis can exert a beneficial effect on organelle homeostasis.

Loss of protein synthesis quality control in host-restricted organisms

Intracellular organisms, such as obligate parasites and endosymbionts, typically possess small genomes due to continuous genome decay caused by an environment with alleviated natural selection. Previously, a few species with highly reduced genomes, including the intracellular pathogens Mycoplasma and Microsporidia, have been shown to carry degenerated editing domains in aminoacyl-tRNA synthetases. These defects in the protein synthesis machinery cause inaccurate translation of the genetic code, resulting in significant statistical errors in protein sequences that are thought to help parasites to escape immune response of a host. In this study we analyzed 10,423 complete bacterial genomes to assess conservation of the editing domains in tRNA synthetases, including LeuRS, IleRS, ValRS, ThrRS, AlaRS, and PheRS. We found that, while the editing domains remain intact in free-living species, they are degenerated in the overwhelming majority of host-restricted bacteria. Our work illustrates that massive genome erosion triggered by an intracellular lifestyle eradicates one of the most fundamental components of a living cell: the system responsible for proofreading of amino acid selection for protein synthesis. This finding suggests that inaccurate translation of the genetic code might be a general phenomenon among intercellular organisms with reduced genomes.

Distinct types of translation termination generate substrates for ribosome-associated quality control.

Cotranslational degradation of polypeptide nascent chains plays a critical role in quality control of protein synthesis and the rescue of stalled ribosomes. In eukaryotes, ribosome stalling triggers release of 60S subunits with attached nascent polypeptides, which undergo ubiquitination by the E3 ligase Ltn1 and proteasomal degradation facilitated by the ATPase Cdc48. However, the identity of factors acting upstream in this process is less clear. Here, we examined how the canonical release factors Sup45–Sup35 (eRF1–eRF3) and their paralogs Dom34-Hbs1 affect the total population of ubiquitinated nascent chains associated with yeast ribosomes. We found that the availability of the functional release factor complex Sup45–Sup35 strongly influences the amount of ubiquitinated polypeptides associated with 60S ribosomal subunits, while Dom34-Hbs1 generate 60S-associated peptidyl-tRNAs that constitute a relatively minor fraction of Ltn1 substrates. These results uncover two separate pathways that target nascent polypeptides for Ltn1-Cdc48-mediated degradation and suggest that in addition to canonical termination on stop codons, eukaryotic release factors contribute to cotranslational protein quality control.

Relaxed substrate specificity leads to extensive tRNA mischarging by Streptococcus pneumoniae class I and class II aminoacyl-tRNA synthetases.

ABSTRACTAminoacyl-tRNA synthetases provide the first step in protein synthesis quality control by discriminating cognate from noncognate amino acid and tRNA substrates. While substrate specificity is enhanced in many instances by cis- and trans-editing pathways, it has been revealed that in organisms such as Streptococcus pneumoniae some aminoacyl-tRNA synthetases display significant tRNA mischarging activity. To investigate the extent of tRNA mischarging in this pathogen, the aminoacylation profiles of class I isoleucyl-tRNA synthetase (IleRS) and class II lysyl-tRNA synthetase (LysRS) were determined. Pneumococcal IleRS mischarged tRNAIle with both Val, as demonstrated in other bacteria, and Leu in a tRNA sequence-dependent manner. IleRS substrate specificity was achieved in an editing-independent manner, indicating that tRNA mischarging would only be significant under growth conditions where Ile is depleted. Pneumococcal LysRS was found to misaminoacylate tRNALys with Ala and to a lesser extent Thr and Ser, with mischarging efficiency modulated by the presence of an unusual U4:G69 wobble pair in the acceptor stems of both pneumococcal tRNALys isoacceptors. Addition of the trans-editing factor MurM, which also functions in peptidoglycan synthesis, reduced Ala-tRNALys production by LysRS, providing evidence for cross talk between the protein synthesis and cell wall biogenesis pathways. Mischarging of tRNALys by AlaRS was also observed, and this would provide additional potential MurM substrates. More broadly, the extensive mischarging activities now described for a number of Streptococcus pneumoniae aminoacyl-tRNA synthetases suggest that adaptive misaminoacylation may contribute significantly to the viability of this pathogen during amino acid starvation.

OASIS regulates chondroitin 6‐O‐sulfotransferase 1 gene transcription in the injured adult mouse cerebral cortex

Old astrocyte specifically induced substance (OASIS), a basic leucine zipper transcription factor of the cAMP response element binding/Activating transcription factor family, is induced in reactive astrocytes in vivo and has important roles in quality control of protein synthesis at the endoplasmic reticulum. Reactive astrocytes produce a non-permissive environment for regenerating axons by up-regulating chondroitin sulfate proteoglycans (CSPGs). In this study, we focus on the potential role of OASIS in CSPG production in the adult mouse cerebral cortex. CS-C immunoreactivity, which represents chondroitin sulfate moieties, was significantly attenuated in the stab-injured cortices of OASIS knockout mice compared to those of wild-type mice. We next examined expression of the CSPG-synthesizing enzymes and core proteins of CSPGs in the stab-injured cortices of OASIS knockout and wild-type mice. The levels of chondroitin 6-O-sulfotransferase 1 (C6ST1, one of the major enzymes involved in sulfation of CSPGs) mRNA and protein increased after cortical stab injury of wild-type, but not of OASIS knockout, mice. A C-terminal deletion mutant OASIS over-expressed in rat C6 glioma cells increased C6ST1 transcription by interacting with the first intron region. Neurite outgrowth of cultured hippocampal neurons was inhibited on culture dishes coated with membrane fractions of epidermal growth factor-treated astrocytes derived from wild type but not from OASIS knockout mice. These results suggest that OASIS regulates the transcription of C6ST1 and thereby promotes CSPG sulfation in astrocytes. Through these mechanisms, OASIS may modulate axonal regeneration in the injured cerebral cortex. OASIS, an ER stress-responsive CREB/ATF family member, is up-regulated in the reactive astrocytes of the injured brain. We found that the up-regulated OASIS is involved in the transcriptional regulation of C6ST1 gene, which promotes chondroitin sulfate proteoglycan (CSPG) sulfation. We conclude that OASIS functions as an anti-regenerative transcription factor by establishing a non-permissive microenvironment to regenerating axons.

Omnipotent role of archaeal elongation factor 1 alpha (EF1α) in translational elongation and termination, and quality control of protein synthesis

The molecular mechanisms of translation termination and mRNA surveillance in archaea remain unclear. In eukaryotes, eRF3 and HBS1, which are homologous to the tRNA carrier GTPase EF1α, respectively bind eRF1 and Pelota to decipher stop codons or to facilitate mRNA surveillance. However, genome-wide searches of archaea have failed to detect any orthologs to both GTPases. Here, we report the crystal structure of aRF1 from an archaeon, Aeropyrum pernix, and present strong evidence that the authentic archaeal EF1α acts as a carrier GTPase for aRF1 and for aPelota. The binding interface residues between aRF1 and aEF1α predicted from aRF1·aEF1α·GTP ternary structure model were confirmed by in vivo functional assays. The aRF1/eRF1 structural domain with GGQ motif, which corresponds to the CCA arm of tRNA, contacts with all three structural domains of aEF1α showing striking tRNA mimicry of aRF1/eRF1 and its GTPase-mediated catalysis for stop codon decoding. The multiple binding capacity of archaeal EF1α explains the absence of GTPase orthologs for eRF3 and HBS1 in archaea species and suggests that universal molecular mechanisms underlie translational elongation and termination, and mRNA surveillance pathways.

Genetic Analysis of the Structure and Function of Transfer Messenger RNA Pseudoknot 1

tmRNA rescues stalled ribosomes in eubacteria by forcing the ribosome to abandon its mRNA template and resume translation with tmRNA itself as a template. Pseudoknot 1 (pk1), immediately upstream of this coding region in tmRNA, is a structural element that is considered essential for tmRNA function based on the analysis of pk1 mutants in vitro. pk1 binds near the ribosomal decoding site and may make base-specific contacts with tmRNA ligands. To study pk1 structure and function in vivo, we have developed a genetic selection that ties the life of Escherichia coli cells to tmRNA activity. Mutation of pk1 at 20% per base and selection for tmRNA activity yielded sequences that retain the same pseudoknot fold. In contrast, selection of active mutants from 10(6) completely random sequences identified hairpin structures that functionally replace pk1. Rational design of a hairpin with increased stability using an unrelated sequence yielded a tmRNA mutant with nearly wild-type activity. We conclude that the role of pk1 in tmRNA function is purely structural and that it can be replaced with a variety of hairpin structures. Our results demonstrate that in the study of functional RNAs, the inactivity of a mutant designed to destroy a given structure should not be interpreted as proof that the structure is necessary for RNA function. Such mutations may only destabilize a global fold that could be formed equally well by an entirely different, stable structure.

Loss of Editing Activity during the Evolution of Mitochondrial Phenylalanyl-tRNA Synthetase

Accurate selection of amino acids is essential for faithful translation of the genetic code. Errors during amino acid selection are usually corrected by the editing activity of aminoacyl-tRNA synthetases such as phenylalanyl-tRNA synthetases (PheRS), which edit misactivated tyrosine. Comparison of cytosolic and mitochondrial PheRS from the yeast Saccharomyces cerevisiae suggested that the organellar protein might lack the editing activity. Yeast cytosolic PheRS was found to contain an editing site, which upon disruption abolished both cis and trans editing of Tyr-tRNA(Phe). Wild-type mitochondrial PheRS lacked cis and trans editing and could synthesize Tyr-tRNA(Phe), an activity enhanced in active site variants with improved tyrosine recognition. Possible trans editing was investigated in isolated mitochondrial extracts, but no such activity was detected. These data indicate that the mitochondrial protein synthesis machinery lacks the tyrosine proofreading activity characteristic of cytosolic translation. This difference between the mitochondria and the cytosol suggests that either organellar protein synthesis quality control is focused on another step or that translation in this compartment is inherently less accurate than in the cytosol.

A freestanding proofreading domain is required for protein synthesis quality control in Archaea.

Threonyl-tRNA synthetase (ThrRS) participates in protein synthesis quality control by selectively editing the misacylated species Ser-tRNA(Thr). In bacteria and eukaryotes the editing function of ThrRS resides in a highly conserved N-terminal domain distant from the active site. Most archaeal ThrRS proteins are devoid of this editing domain, suggesting evolutionary divergence of quality-control mechanisms. Here we show that archaeal editing of Ser-tRNAThr is catalyzed by a domain unrelated to, and absent from, bacterial and eukaryotic ThrRSs. Despite the lack of sequence homology, the archaeal and bacterial editing domains are both reliant on a pair of essential histidine residues suggestive of a common catalytic mechanism. Whereas the archaeal editing module is most commonly part of full-length ThrRS, several crenarchaeal species contain individual genes encoding the catalytic (ThrRS-cat) and editing domains (ThrRS-ed). Sulfolobus solfataricus ThrRS-cat was shown to synthesize both Thr-tRNAThr and Ser-tRNAThr and to lack editing activity against Ser-tRNAThr. In contrast, ThrRS-ed lacks aminoacylation activity but can act as an autonomous protein in trans to hydrolyze specifically Ser-tRNAThr, or it can be fused to ThrRS-cat to provide the same function in cis. Deletion analyses indicate that ThrRS-ed is dispensable for growth of S. solfataricus under standard conditions but is required for normal growth in media with elevated serine levels. The growth phenotype of the ThrRS-ed deletion strain suggests that retention of the discontinuous ThrRS quaternary structure relates to specific physiological requirements still evident in certain Archaea.

Ribosome Stalling during Translation Elongation Induces Cleavage of mRNA Being Translated in Escherichia coli

Recently, it has been found that ribosome pausing at stop codons caused by certain nascent peptides induces cleavage of mRNA in Escherichia coli cells (1, 2). The question we addressed in the present study is whether mRNA cleavage occurs when translation elongation is prevented. We focused on a specific peptide sequence (AS17), derived from SecM, that is known to cause elongation arrest. When the crp-crr fusion gene encoding CRP-AS17-IIA(Glc) was expressed, cAMP receptor protein (CRP) proteins truncated around the arrest sequence were efficiently produced, and they were tagged by the transfer-messenger RNA (tmRNA) system. Northern blot analysis revealed that both truncated upstream crp and downstream crr mRNAs were generated along with reduced amounts of the full-length crp-crr mRNA. The truncated crp mRNA dramatically decreased in the presence of tmRNA due to rapid degradation. The 3' ends of truncated crp mRNA correspond well to the C termini of the truncated CRP proteins. We conclude that ribosome stalling by the arrest sequence induces mRNA cleavage near the arrest point, resulting in nonstop mRNAs that are recognized by tmRNA. We propose that the mRNA cleavage induced by ribosome stalling acts in concert with the tmRNA system as a way to ensure quality control of protein synthesis and possibly to regulate the expression of certain genes.