Translation termination in pyrrolysine-utilizing archaea.
Ontology highlight
ABSTRACT:
SUBMITTER: Alkalaeva E
PROVIDER: S-EPMC2857517 | biostudies-literature | 2009 Nov
REPOSITORIES: biostudies-literature
Publications
Translation termination in pyrrolysine-utilizing archaea.
Alkalaeva Elena E Eliseev Boris B Ambrogelly Alexandre A Vlasov Peter P Kondrashov Fyodor A FA Gundllapalli Sharath S Frolova Lyudmila L Söll Dieter D Kisselev Lev L
FEBS letters 20090929 21
Although some data link archaeal and eukaryotic translation, the overall mechanism of protein synthesis in archaea remains largely obscure. Both archaeal (aRF1) and eukaryotic (eRF1) single release factors recognize all three stop codons. The archaeal genus Methanosarcinaceae contains two aRF1 homologs, and also uses the UAG stop to encode the 22nd amino acid, pyrrolysine. Here we provide an analysis of the last stage of archaeal translation in pyrrolysine-utilizing species. We demonstrated that ...[more]
Similar Datasets
Project description:The pyrrolysyl-tRNA synthetase (PylRS) facilitates the cotranslational installation of the 22nd amino acid pyrrolysine. Owing to its tolerance for diverse amino acid substrates, and its orthogonality in multiple organisms, PylRS has emerged as a major route to install noncanonical amino acids into proteins in living cells. Recently, a novel class of PylRS enzymes was identified in a subset of methanogenic archaea. Enzymes within this class (ΔPylSn) lack the N-terminal tRNA-binding domain that is widely conserved amongst PylRS enzymes, yet remain active and orthogonal in bacteria and eukaryotes. In this study, we use biochemical and in vivo UAG-readthrough assays to characterize the aminoacylation efficiency and substrate spectrum of a ΔPylSn class PylRS from the archaeon Candidatus Methanomethylophilus alvus. We show that, compared with the full-length enzyme from Methanosarcina mazei, the Ca. M. alvus PylRS displays reduced aminoacylation efficiency but an expanded amino acid substrate spectrum. To gain insight into the evolution of ΔPylSn enzymes, we performed molecular phylogeny using 156 PylRS and 105 pyrrolysine tRNA (tRNAPyl) sequences from diverse archaea and bacteria. This analysis suggests that the PylRS•tRNAPyl pair diverged before the evolution of the three domains of life, placing an early limit on the evolution of the Pyl-decoding trait. Furthermore, our results document the coevolutionary history of PylRS and tRNAPyl and reveal the emergence of tRNAPyl sequences with unique A73 and U73 discriminator bases. The orthogonality of these tRNAPyl species with the more common G73-containing tRNAPyl will enable future efforts to engineer PylRS systems for further genetic code expansion.
| S-EPMC9630628 | biostudies-literature
Project description:The genomes of many prokaryotes contain substantial fractions of gene pairs with overlapping stop and start codons (ATGA or TGATG). A potential benefit of overlapping gene pairs is translational coupling. In 720 genomes of archaea and bacteria representing all major phyla, we identify substantial, albeit highly variable, fractions of co-directed overlapping gene pairs. Various patterns are observed for the utilization of the SD motif for de novo initiation at upstream genes versus reinitiation at overlapping gene pairs. We experimentally test the predicted coupling in 9 gene pairs from the archaeon Haloferax volcanii and 5 gene pairs from the bacterium Escherichia coli. In 13 of 14 cases, translation of both genes is strictly coupled. Mutational analysis of SD motifs located upstream of the downstream genes indicate that the contribution of the SD to translational coupling widely varies from gene to gene. The nearly universal, abundant occurrence of overlapping gene pairs suggests that tight translational coupling is widespread in archaea and bacteria.
| S-EPMC6728339 | biostudies-literature
Project description:In contrast to bacteria that have two release factors, RF1 and RF2, eukaryotes only possess one unrelated release factor eRF1, which recognizes all three stop codons of the mRNA and hydrolyses the peptidyl-tRNA bond. While the molecular basis for bacterial termination has been elucidated, high-resolution structures of eukaryotic termination complexes have been lacking. Here we present a 3.8 Å structure of a human translation termination complex with eRF1 decoding a UAA(A) stop codon. The complex was formed using the human cytomegalovirus (hCMV) stalling peptide, which perturbs the peptidyltransferase center (PTC) to silence the hydrolysis activity of eRF1. Moreover, unlike sense codons or bacterial stop codons, the UAA stop codon adopts a U-turn-like conformation within a pocket formed by eRF1 and the ribosome. Inducing the U-turn conformation for stop codon recognition rationalizes how decoding by eRF1 includes monitoring geometry in order to discriminate against sense codons.
| S-EPMC4605324 | biostudies-literature
Project description:Termination of mRNA translation occurs when a stop codon enters the A site of the ribosome, and in eukaryotes is mediated by release factors eRF1 and eRF3, which form a ternary eRF1/eRF3-guanosine triphosphate (GTP) complex. eRF1 recognizes the stop codon, and after hydrolysis of GTP by eRF3, mediates release of the nascent peptide. The post-termination complex is then disassembled, enabling its constituents to participate in further rounds of translation. Ribosome recycling involves splitting of the 80S ribosome by the ATP-binding cassette protein ABCE1 to release the 60S subunit. Subsequent dissociation of deacylated transfer RNA (tRNA) and messenger RNA (mRNA) from the 40S subunit may be mediated by initiation factors (priming the 40S subunit for initiation), by ligatin (eIF2D) or by density-regulated protein (DENR) and multiple copies in T-cell lymphoma-1 (MCT1). These events may be subverted by suppression of termination (yielding carboxy-terminally extended read-through polypeptides) or by interruption of recycling, leading to reinitiation of translation near the stop codon.
| S-EPMC6169810 | biostudies-literature
Project description:Mutations that introduce premature termination codons (PTCs) within protein-coding genes are associated with incurable and severe genetic diseases. Many PTC-associated disorders are life-threatening and have no approved medical treatment options. Suppressor transfer RNAs (sup-tRNAs) with the capacity to promote translational readthrough of PTCs represent a promising therapeutic strategy to treat these conditions; however, developing novel sup-tRNAs with high efficiency and specificity often requires extensive engineering and screening. Moreover, these efforts are not always successful at producing more efficient sup-tRNAs. Here we show that a pyrrolysine tRNA (tRNAPyl), which naturally translates the UAG stop codon, offers an attractive scaffold for developing effective sup-tRNAs that restore protein synthesis from PTC-containing genes. We created a series of rationally designed Pyrrolysine tRNA Scaffold Suppressor-tRNAs (PASS-tRNAs) that are substrates of bacterial and human alanyl-tRNA synthetase. Using a PTC-containing fluorescent reporter gene, PASS-tRNAs restore protein synthesis to wild-type levels in bacterial cells. In human cells, PASS-tRNAs display robust and consistent PTC suppression in multiple reporter genes, including pathogenic mutations in the tumor suppressor gene BRCA1 associated with breast and ovarian cancer. Moreover, PTC suppression occurred with high codon specificity and no observed cytotoxic effects. Collectively, these results unveil a class of sup-tRNAs with great potential for tRNA-based therapeutics.
2024-12-27 | GSE277128 | GEO
Project description:BACKGROUND:A seventh order of methanogens, the Methanomassiliicoccales, has been identified in diverse anaerobic environments including the gastrointestinal tracts (GIT) of humans and other animals and may contribute significantly to methane emission and global warming. Methanomassiliicoccales are phylogenetically distant from all other orders of methanogens and belong to a large evolutionary branch composed by lineages of non-methanogenic archaea such as Thermoplasmatales, the Deep Hydrothermal Vent Euryarchaeota-2 (DHVE-2, Aciduliprofundum boonei) and the Marine Group-II (MG-II). To better understand this new order and its relationship to other archaea, we manually curated and extensively compared the genome sequences of three Methanomassiliicoccales representatives derived from human GIT microbiota, "Candidatus Methanomethylophilus alvus", "Candidatus Methanomassiliicoccus intestinalis" and Methanomassiliicoccus luminyensis. RESULTS:Comparative analyses revealed atypical features, such as the scattering of the ribosomal RNA genes in the genome and the absence of eukaryotic-like histone gene otherwise present in most of Euryarchaeota genomes. Previously identified in Thermoplasmatales genomes, these features are presently extended to several completely sequenced genomes of this large evolutionary branch, including MG-II and DHVE2. The three Methanomassiliicoccales genomes share a unique composition of genes involved in energy conservation suggesting an original combination of two main energy conservation processes previously described in other methanogens. They also display substantial differences with each other, such as their codon usage, the nature and origin of their CRISPRs systems and the genes possibly involved in particular environmental adaptations. The genome of M. luminyensis encodes several features to thrive in soil and sediment conditions suggesting its larger environmental distribution than GIT. Conversely, "Ca. M. alvus" and "Ca. M. intestinalis" do not present these features and could be more restricted and specialized on GIT. Prediction of the amber codon usage, either as a termination signal of translation or coding for pyrrolysine revealed contrasted patterns among the three genomes and suggests a different handling of the Pyl-encoding capacity. CONCLUSIONS:This study represents the first insights into the genomic organization and metabolic traits of the seventh order of methanogens. It suggests contrasted evolutionary history among the three analyzed Methanomassiliicoccales representatives and provides information on conserved characteristics among the overall methanogens and among Thermoplasmata.
| S-EPMC4153887 | biostudies-literature
Project description:Characterizing the structural dynamics of the translating ribosome remains a major goal in the study of protein synthesis. Deacylation of peptidyl-tRNA during translation elongation triggers fluctuations of the pretranslocation ribosomal complex between two global conformational states. Elongation factor G-mediated control of the resulting dynamic conformational equilibrium helps to coordinate ribosome and tRNA movements during elongation and is thus a crucial mechanistic feature of translation. Beyond elongation, deacylation of peptidyl-tRNA also occurs during translation termination, and this deacylated tRNA persists during ribosome recycling. Here we report that specific regulation of the analogous conformational equilibrium by translation release and ribosome recycling factors has a critical role in the termination and recycling mechanisms. Our results support the view that specific regulation of the global state of the ribosome is a fundamental characteristic of all translation factors and a unifying theme throughout protein synthesis.
| S-EPMC4748396 | biostudies-literature
Project description:The bacterial tmRNA·SmpB system facilitates recycling of stalled translational complexes in a process termed "ribosome rescue." During ribosome rescue, the nascent chain is tagged with the tmRNA-encoded ssrA peptide, which targets the tagged polypeptide for degradation. Translational pausing also induces a variety of recoding events such as frameshifts, ribosome hops, and stop codon readthrough. To examine the interplay between recoding and ribosome rescue, we determined the various fates of ribosomes that pause during translation termination. We expressed a model protein containing the C-terminal Asp-Pro nascent peptide motif (which interferes with translation termination) and quantified the protein chains produced by recoding and ssrA-peptide tagging. The nature and extent of translational recoding depended upon the codon for the C-terminal Pro residue, with CCU and CCC promoting efficient +1 frameshifting. In contrast, ssrA-peptide tagging was unaffected by C-terminal Pro coding. Moreover, +1 frameshifting was not suppressed by tmRNA·SmpB activity, suggesting that recoding and ribosome rescue are not competing events. However, cells lacking ribosomal protein L9 (?L9) exhibited a significant increase in recoding and a concomitant decrease in ssrA-peptide tagging. Pulse-chase analysis revealed that pre-termination ribosomes turn over more rapidly in ?L9 cells, suggesting that increased recoding alleviates the translational arrest. Together, these results indicate that tmRNA·SmpB does not suppress transient ribosome pauses, but responds to prolonged translational arrest.
| S-EPMC3173060 | biostudies-literature
Project description:Terminating protein translation accurately and efficiently is critical for both protein fidelity and ribosome recycling for continued translation. The three bacterial release factors (RFs) play key roles: RF1 and 2 recognize stop codons and terminate translation; and RF3 promotes disassociation of bound release factors. Probing release factors mutations with reporter constructs containing programmed frameshifting sequences or premature stop codons had revealed a propensity for readthrough or frameshifting at these specific sites, but their effects on translation genome-wide have not been examined. We performed ribosome profiling on a set of isogenic strains with well-characterized release factor mutations to determine how they alter translation globally. Consistent with their known defects, strains with increasingly severe release factor defects exhibit increasingly severe accumulation of ribosomes over stop codons, indicative of an increased duration of the termination/release phase of translation. Release factor mutant strains also exhibit increased occupancy in the region following the stop codon at a significant number of genes. Our global analysis revealed that, as expected, translation termination is generally efficient and accurate, but that at a significant number of genes (≥ 50) the ribosome signature after the stop codon is suggestive of translation past the stop codon. Even native E. coli K-12 exhibits the ribosome signature suggestive of protein extension, especially at UGA codons, which rely exclusively on the reduced function RF2 variant of the K-12 strain for termination. Deletion of RF3 increases the severity of the defect. We unambiguously demonstrate readthrough and frameshifting protein extensions and their further accumulation in mutant strains for a few select cases. In addition to enhancing recoding, ribosome accumulation over stop codons disrupts attenuation control of biosynthetic operons, and may alter expression of some overlapping genes. Together, these functional alterations may either augment the protein repertoire or produce deleterious proteins.
| S-EPMC5373646 | biostudies-literature
Project description:The eukaryotic translation factor eIF5A, originally identified as an initiation factor, was later shown to promote translation elongation of iterated proline sequences. Using a combination of ribosome profiling and in vitro biochemistry, we report a much broader role for eIF5A in elongation and uncover a critical function for eIF5A in termination. Ribosome profiling of an eIF5A-depleted strain reveals a global elongation defect, with abundant ribosomes stalling at many sequences, not limited to proline stretches. Our data also show ribosome accumulation at stop codons and in the 3' UTR, suggesting a global defect in termination in the absence of eIF5A. Using an in vitro reconstituted translation system, we find that eIF5A strongly promotes the translation of the stalling sequences identified by profiling and increases the rate of peptidyl-tRNA hydrolysis more than 17-fold. We conclude that eIF5A functions broadly in elongation and termination, rationalizing its high cellular abundance and essential nature.
| S-EPMC5414311 | biostudies-literature