Like all other secretory proteins, the HIV-1 envelope glycoprotein gp160 is targeted to the endoplasmic reticulum (ER) by its signal peptide during synthesis. Proper gp160 folding in the ER requires core glycosylation, disulfide-bond formation and proline isomerization. Signal-peptide cleavage occurs only late after gp160 chain termination and is dependent on folding of the soluble subunit gp120 to a near-native conformation. We here detail the mechanism by which co-translational signal-peptide cleavage is prevented. Conserved residues from the signal peptide and residues downstream of the canonical cleavage site form an extended alpha-helix in the ER membrane, which covers the cleavage site, thus preventing cleavage. A point mutation in the signal peptide breaks the alpha helix allowing co-translational cleavage. We demonstrate that postponed cleavage of gp160 enhances functional folding of the molecule. The change to early cleavage results in decreased viral fitness compared to wild-type HIV.

Bypassing is a recoding event that leads to the translation of two distal open reading frames into a single polypeptide chain. We present the structure of a translating ribosome stalled at the bypassing take-off site of gene 60 of bacteriophage T4. The nascent peptide in the exit tunnel anchors the P-site peptidyl-tRNAGly to the ribosome and locks an inactive conformation of the peptidyl transferase center (PTC). The mRNA forms a short dynamic hairpin in the decoding site. The ribosomal subunits adopt a rolling conformation in which the rotation of the small subunit around its long axis causes the opening of the A-site region. Together, PTC conformation and mRNA structure safeguard against premature termination and read-through of the stop codon and reconfigure the ribosome to a state poised for take-off and sliding along the noncoding mRNA gap.

Molecular chaperones assist with protein folding by interacting with nascent polypeptide chains (NCs) during translation. Whether the ribosome can sense chaperone defects and, in response, abort translation of misfolding NCs has not yet been explored. Here we used quantitative proteomics to investigate the ribosome-associated chaperone network in E. coli and the consequences of its dysfunction. Trigger factor and the DnaK (Hsp70) system are the major NC-binding chaperones. HtpG (Hsp90), GroEL, and ClpB contribute increasingly when DnaK is deficient. Surprisingly, misfolding because of defects in co-translational chaperone function or amino acid analog incorporation results in recruitment of the non-canonical release factor RF3. RF3 recognizes aberrant NCs and then moves to the peptidyltransferase site to cooperate with RF2 in mediating chain termination, facilitating clearance by degradation. This function of RF3 reduces the accumulation of misfolded proteins and is critical for proteostasis maintenance and cell survival under conditions of limited chaperone availability.

Expression of viral proteins frequently includes non-canonical decoding events ('recoding') during translation. '2A' oligopeptides drive one such event, termed 'stop-carry on' recoding. Nascent 2A peptides interact with the ribosomal exit tunnel to dictate an unusual stop codon-independent termination of translation at the final Pro codon of 2A. Subsequently, translation 'reinitiates' on the same codon, two individual proteins being generated from one open reading frame. Many 2A peptides have been identified, and they have a conserved C-terminal motif. Little similarity is present in the N-terminal portions of these peptides, which might suggest that these amino acids are not important in the 2A reaction. However, mutagenesis indicates that identity of the amino acid at nearly all positions of a single 2A peptide is important for activity. Each 2A may then represent a specific solution for positioning the conserved C-terminus within the peptidyl-transferase centre to promote recoding. Nascent 2A peptide:ribosome interactions are suggested to alter ribosomal fine structure to discriminate against prolyl-tRNA(Pro) and promote termination in the absence of a stop codon. Such structural modifications may account for our observation that replacement of the final Pro codon of 2A with any stop codon both stalls ribosome processivity and inhibits nascent chain release.

The malaria parasite Plasmodium spp. varies the expression profile of its genes depending on the host it resides in and its developmental stage. Virtually all messenger RNA (mRNA) is expressed in a monocistronic manner, with transcriptional activation regulated at the epigenetic level and by specialized transcription factors. Furthermore, recent systems-wide studies have identified distinct mechanisms of post-transcriptional and translational control at various points of the parasite lifecycle. Taken together, it is evident that 'just-in-time' transcription and translation strategies coexist and coordinate protein expression during Plasmodium development, some of which we review here. In particular, we discuss global and specific mechanisms that control protein translation in blood stages of the human malaria parasite Plasmodium falciparum, once a cytoplasmic mRNA has been generated, and its crosstalk with mRNA decay and storage. We also focus on the widespread translational delay observed during the 48-hour blood stage lifecycle of P. falciparum-for over 30% of transcribed genes, including virulence factors required to invade erythrocytes-and its regulation by cis-elements in the mRNA, RNA-processing enzymes and RNA-binding proteins; the first-characterized amongst these are the DNA- and RNA-binding Alba proteins. More generally, we conclude that translational regulation is an emerging research field in malaria parasites and propose that its elucidation will not only shed light on the complex developmental program of this parasite, but may also reveal mechanisms contributing to drug resistance and define new targets for malaria intervention strategies. WIREs RNA 2016, 7:772-792. doi: 10.1002/wrna.1365 For further resources related to this article, please visit the WIREs website.

In Escherichia coli, prolonged translational arrest allows mRNA degradation into the A site of stalled ribosomes. The enzyme that cleaves the A-site codon is not known, but its activity requires RNase II to degrade mRNA downstream of the ribosome. This A-site mRNA cleavage process is thought to function in translation quality control because stalled ribosomes are recycled from A-site truncated transcripts by the tmRNA-SmpB "ribosome rescue" system. During rescue, the tmRNA-encoded ssrA peptide is added to the nascent chain, thereby targeting the tagged protein for degradation after release from the ribosome. Here, we examine the influence of A-site mRNA cleavage upon tmRNA-SmpB activity. Using a model transcript that undergoes stop-codon cleavage in response to inefficient translation termination, we quantify ssrA-peptide tagging of the encoded protein in cells that contain (rnb(+)) or lack (Δrnb) RNase II. A-site mRNA cleavage is reduced approximately three-fold in Δrnb backgrounds, but the efficiency of ssrA-tagging is identical to that of rnb(+) cells. Additionally, pulse-chase analysis demonstrates that paused ribosomes recycle from the test transcripts at similar rates in rnb(+) and Δrnb cells. Together, these results indicate that A-site truncated transcripts are not required for tmRNA-SmpB-mediated ribosome rescue and suggest that A-site mRNA cleavage process may play a role in other recycling pathways.

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.

Translation termination in eukaryotes is governed by the concerted action of eRF1 and eRF3 factors. eRF1 recognizes the stop codon in the A site of the ribosome and promotes nascent peptide chain release, and the GTPase eRF3 facilitates this peptide release via its interaction with eRF1. In addition to its role in termination, eRF3 is involved in normal and nonsense-mediated mRNA decay through its association with cytoplasmic poly(A)-binding protein (PABP) via PAM2-1 and PAM2-2 motifs in the N-terminal domain of eRF3. We have studied complex formation between full-length eRF3 and its ligands (GDP, GTP, eRF1 and PABP) using isothermal titration calorimetry, demonstrating formation of the eRF1:eRF3:PABP:GTP complex. Analysis of the temperature dependence of eRF3 interactions with G nucleotides reveals major structural rearrangements accompanying formation of the eRF1:eRF3:GTP complex. This is in contrast to eRF1:eRF3:GDP complex formation, where no such rearrangements were detected. Thus, our results agree with the established active role of GTP in promoting translation termination. Through point mutagenesis of PAM2-1 and PAM2-2 motifs in eRF3, we demonstrate that PAM2-2, but not PAM2-1 is indispensible for eRF3:PABP complex formation.

Translational stalling of ribosome bound to endoplasmic reticulum (ER) membrane requires an accurate clearance of the associated polypeptides, which is not completely understood in mammals. We characterized in mammalian cells the model of ribosomal stalling at the STOP-codon based on proteins tagged at the C-terminus with the picornavirus 2A peptide followed by a termination codon instead of the Proline (2A*). We exploited the 2A* stalling model to characterize the pathway of degradation of ER-targeted polypeptides. We report that the ER chaperone BiP/GRP78 is a new main factor involved. Moreover, degradation of the ER-stalled polypeptides required the activities of the AAA-ATPase VCP/p97, its associated deubiquitinylase YOD1, the ribosome-associated ubiquitin ligase Listerin and the proteasome. In human proteome, we found two human C-terminal amino acid sequences that cause similar stalling at the STOP-codon. Our data suggest that translational stalling at the ER membrane activates protein degradation at the interface of ribosomal- and ER-associated quality control systems.

Basket clam soup, a popular Asian dish, is prepared by boiling clams in hot water. The soup is generally cloudy, and it is considered that increased cloudiness enhances taste. However, the composition of the whitening ingredients and their association with taste enhancement remains unclear. In this study, we aimed to identify the components contributing to the white colour of the boiled soup. The white component upon precipitation with trichloroacetic acid reacted positively with ninhydrin, indicating the presence of proteins. The separation of proteins using sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed an intense band of size 33 kDa. Peptide mass fingerprinting of the identified protein using matrix-assisted laser desorption/ionisation-time-of-flight tandem mass spectrometry revealed the protein as tropomyosin. To validate the involvement of tropomyosin in the turbidity of the soup, tropomyosin was expressed and extracted from Escherichia coli. As expected, the purified protein suspended in water resulted in turbid appearance. To determine whether lipids have any association with the observed cloudiness of the soup, the amounts of fatty acids were measured. The proportion of estimated fatty acids was very low compared to that of proteins. Overall, we identified the major component contributing to soup cloudiness as tropomyosin forming micelles.

Despite limited reports on glutamine methylation, methylated glutamine is found to be highly conserved in a "GGQ" motif in both prokaryotes and eukaryotes. In bacteria, glutamine methylation of peptide chain release factors 1/2 (RF1/2) by the enzyme PrmC is essential for translational termination and transcript recycling. Two PrmC homologs, HEMK1 and HEMK2, are found in mammals. In contrast to those of HEMK2, the biochemical properties and biological significance of HEMK1 remain largely unknown. In this study, we demonstrated that HEMK1 is an active methyltransferase for the glutamine residue of the GGQ motif of all four putative mitochondrial release factors (mtRFs)-MTRF1, MTRF1L, MRPL58, and MTRFR. In HEMK1-deficient HeLa cells, GGQ motif glutamine methylation was absent in all the mtRFs. We examined cell growth and mitochondrial properties, but disruption of the HEMK1 gene had no considerable impact on the overall cell growth, mtDNA copy number, mitochondrial membrane potential, and mitochondrial protein synthesis under regular culture condition with glucose as a carbon source. Furthermore, cell growth potential of HEMK1 KO cells was still maintained in the respiratory condition with galactose medium. Our results suggest that HEMK1 mediates the GGQ methylation of all four mtRFs in human cells; however, this specific modification seems mostly dispensable in cell growth and mitochondrial protein homeostasis at least for HeLa cells under fermentative culture condition.

The Burkholderia genus possesses ecological and metabolic diversities. A large number of silent biosynthetic gene clusters (BGCs) in the Burkholderia genome remain uncharacterized and represent a promising resource for new natural product discovery. However, exploitation of the metabolomic potential of Burkholderia is limited by the absence of efficient genetic manipulation tools. Here, we screened a bacteriophage recombinase system Redγ-BAS, which was functional for genome modification in the plant pathogen Burkholderia gladioli ATCC 10248. By using this recombineering tool, the constitutive promoters were precisely inserted in the genome, leading to activation of two silent nonribosomal peptide synthetase gene clusters (bgdd and hgdd) and production of corresponding new classes of lipopeptides, burriogladiodins A-H (1-8) and haereogladiodins A-B (9-10). Structure elucidation revealed an unnatural amino acid Z- dehydrobutyrine (Dhb) in 1-8 and an E-Dhb in 9-10. Notably, compounds 2-4 and 9 feature an unusual threonine tag that is longer than the predicted collinearity assembly lines. The structural diversity of burriogladiodins was derived from the relaxed substrate specificity of the fifth adenylation domain as well as chain termination conducted by water or threonine. The recombinase-mediating genome editing system is not only applicable in B. gladioli, but also possesses great potential for mining meaningful silent gene clusters from other Burkholderia species.

Human astroviruses are small non-enveloped viruses with positive-sense single-stranded RNA genomes. Astroviruses cause acute gastroenteritis in children worldwide and have been associated with encephalitis and meningitis in immunocompromised individuals. It is still unknown how astrovirus particles exit infected cells following replication. Through comparative genomic analysis and ribosome profiling we here identify and confirm the expression of a conserved alternative-frame ORF, encoding the protein XP. XP-knockout astroviruses are attenuated and pseudo-revert on passaging. Further investigation into the function of XP revealed plasma and trans Golgi network membrane-associated roles in virus assembly and/or release through a viroporin-like activity. XP-knockout replicons have only a minor replication defect, demonstrating the role of XP at late stages of infection. The discovery of XP advances our knowledge of these important human viruses and opens an additional direction of research into their life cycle and pathogenesis.

Classic Ehlers-Danlos syndrome (cEDS) is a rare autosomal dominant connective tissue disorder that is primarily characterized by skin hyperextensibility, abnormal wound healing/atrophic scars, and joint hypermobility. A recent study demonstrated that more than 90% of patients who satisfy all of these major criteria harbor a type V collagen (COLLV) defect.