Assembly and functionality of the ribosome with tethered subunits
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA489275 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8413900.fastq.gz | Fastqsanger.gz | |||
| SRR8413893.fastq.gz | Fastqsanger.gz | |||
| SRR8413894.fastq.gz | Fastqsanger.gz | |||
| SRR8413895.fastq.gz | Fastqsanger.gz | |||
| SRR8413896.fastq.gz | Fastqsanger.gz |
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Project description:Ribo-T is an engineered ribosome whose small and large subunits are tethered together by insertion of circularly permutated 23S rRNA into 16S rRNA [Orelle, C., Carlson, E. D., Szal, T., Florin, T., Jewett, M. C., Mankin, A.S. Protein synthesis by ribosomes with tethered subunits. Nature 524, 119-124 (2015)]. Whether the growth defects are related to problems with Ribo-T functionality is unclear. The translation in Ribo-T cells was examined by ribosome profiling (Ribo-seq) and RNA sequencing (RNA-seq). Besides the control cells carrying dissociable WT ribosome (SQ171), RIBO-T cells are the original SQ171 strain transformed with pRibo-T plasmid and cured of the plasmid carrying wt rRNA that was evolved to have improved growth characteristics. The Ribo-T strain carries two mutations: a nonsense mutation in the Leu22 codon of the ybeX gene and a missense mutation in codon 549 of the rpsA gene encoding ribosomal protein S1. Analysis of Ribo-T performance by ribosome profiling showed a notably higher ribosome occupancy of the start codons and somewhat increased occupancy at the stop codons of many genes, suggesting that subunit tethering specifically impairs the initiation and termination stages of translation.
2019-02-25 | GSE119454 | GEO
Project description:Ribo-T is an engineered ribosome whose small and large subunits are tethered together by linking 16S rRNA and 23S rRNA in a single molecule. Although Ribo-T can support cell proliferation in the absence of wild type ribosomes, Ribo-T cells grow slower than those with wild type ribosomes. Here, we show that cell growth defect is likely explained primarily by slow Ribo-T assembly rather than its imperfect functionality. Ribo-T maturation is stalled at a late assembly stage. Several post-transcriptional rRNA modifications and some ribosomal proteins are underrepresented in the accumulated assembly intermediates and rRNA ends are incompletely trimmed. Ribosome profiling of Ribo-T cells shows no defects in translation elongation but reveals somewhat higher occupancy by Ribo-T of the start codons and to a lesser extent stop codons, suggesting that subunit tethering mildly affects the initiation and termination stages of translation. Understanding limitations of Ribo-T system offers ways for its future development.
| S-EPMC6389949 | biostudies-literature
Project description:Protein complexes are pivotal to most cellular processes. Emerging evidence indicates that pairs of ribosomes ubiquitously drive the synchronized synthesis and assembly of two protein subunits into homodimeric complexes1-5. These observations suggest protein folding mechanisms of general importance enabled by contacts between nascent chains6,7 – which have thus far rather been considered detrimental8,9. However, owing to their dynamic and heterogeneous nature, the folding of interacting nascent chains remains unexplored. Here, we show that co-translational ribosome pairing allows their nascent chains to ‘chaperone each other’, thus enabling the formation of coiled-coil homodimers from subunits that misfold individually. We developed an integrated single-molecule fluorescence and force spectroscopy approach to probe the folding and assembly of two nascent chains extending from nearby ribosomes, using the intermediate filament lamin as a model system. Ribosome proximity in early translation stages was found to be critical: when interactions between nascent chains are inhibited or delayed, they become trapped in stable misfolded states that are no longer assembly-competent. Conversely, early interactions allow the two nascent chains to nucleate native-like quaternary structures that grow in size and stability as translation advances. We conjecture that protein folding mechanisms enabled by ribosome cooperation are more broadly relevant to intermediate filaments and other protein classes.
2025-06-23 | GSE282964 | GEO
Project description:Co-translational ribosome pairing enables native assembly of misfolding-prone subunits
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Project description:In S. cerevisiae, the ribosome assembly factor Reh1 binds to pre-60S subunits at a late stage during their cytoplasmic maturation. Unlike canonical assembly factors, which associate exclusively with pre-60S subunits, we observed that Reh1 sediments with polysomes in addition to free 60S subunits. We therefore investigated the intriguing possibility that Reh1 remains associated with 60S subunits after the release of the anti-association factor Tif6 and after subunit joining. Here, we show that Reh1-bound nascent 60S subunits associate with 40S subunits to form actively translating ribosomes.
2024-01-24 | GSE246163 | GEO
Project description:Folding of newly synthesized proteins to the native state is a major challenge within the crowded cellular environment, since non-productive interactions can lead to misfolding, aggregation and degradation1. Cells cope with this challenge by coupling synthesis with polypeptide folding and by employing molecular chaperones to safeguard folding already cotranslationally2. However, little is known about the final step of folding, the assembly of polypeptides into complexes, although most of the cellular proteome forms oligomeric assemblies3. In prokaryotes, a proof-of-concept study showed that assembly of heterodimeric luciferase is an organized cotranslational process, facilitated by spatially confined translation of the subunits encoded on a polycistronic mRNA4. In eukaryotes, however, fundamental differences such as rarity of polycistronic mRNAs and different chaperone constellations raise the question whether assembly is also coordinated with translation. Here we provide a systematic and mechanistic analysis of protein complex assembly in eukaryotes using ribosome profiling. We determined the in vivo nascent subunits interactions of 12 hetero-oligomeric protein complexes of Saccharomyces cerevisiae at near-residue resolution. We find 9 complexes assemble cotranslationally; the 3 complexes that do not show cotranslational interactions are regulated by dedicated assembly chaperones5-7. Cotranslational assembly often occurs uni-directionally, with one fully synthesized subunit engaging its nascent partner subunit(s), thereby counteracting its aggregation propensity. The onset of cotranslational subunit association coincides sharply with full exposure of the nascent interaction domain at the ribosomal tunnel exit. The action of the ribosome-associated Hsp70 chaperone Ssb8 is coordinated with assembly. Ssb transiently engages partially synthesized interaction domains, then dissociates before the onset of partner subunit association, presumably to prevent premature assembly interactions. Our study shows that cotranslational subunit association is a prevalent mechanism for hetero-oligomers assembly in yeast and indicates that translation, folding and assembly of protein complexes are integrated processes in eukaryotes.
2018-07-05 | GSE116570 | GEO
Project description:we prepared synthetic proteins corresponding to 80 of the ribosome subunits by stable isotope labeling and then performed absolute quantification.
2023-03-16 | PXD034018 | JPOST Repository
Project description:RNA-based macromolecular machines, such as the ribosome, have functional parts reliant on structural interactions spanning sequence-distant regions. These features limit evolutionary exploration of mutant libraries and confound three-dimensional structure-guided design. To address these challenges, we describe Evolink (evolution and linkage), a method that enables high-throughput evolution of sequence-distant regions in large macromolecular machines, and library design guided by computational RNA modeling to enable exploration of structurally stable designs. Using Evolink, we evolved a tethered ribosome with a 58% increased activity in orthogonal protein translation and a 97% improvement in doubling times in SQ171 cells compared to a previously developed tethered ribosome, and reveal new permissible sequences in a pair of ribosomal helices with previously explored biological function. The Evolink approach may enable enhanced engineering of macromolecular machines for new and improved functions for synthetic biology.
| S-EPMC9815830 | biostudies-literature
Project description:In animals the organization of the compact mitochondrial genome and lack of introns have necessitated a unique mechanism for RNA processing. To date the regulation of mitochondrial RNA processing and its importance for ribosome biogenesis and energy metabolism are not clear. To understand the in vivo role of the endoribonuclease component of the RNase P complex, MRPP3, we created conditional knockout mice. Here we show that MRPP3 is essential for life, and heart and skeletal muscle-specific knockout leads to a cardiomyopathy early in life, indicating that it is the only RNase P enzyme in mitochondria. We show that RNA processing is required for the biogenesis of the respiratory chain and mitochondrial function. Transcriptome-wide parallel analyses of RNA ends (PARE) and RNA-Seq enabled us to identify the in vivo cleavage sites of RNase P. Cleavage of the 5â² tRNA ends precedes 3â² end processing in vivo and is required for the correct biogenesis of the mitochondrial ribosomal subunits and mitoribosomal proteins that are differentially stabilized or degraded in the absence of mature rRNAs. Finally we identify that large mitoribosomal proteins can form a subcomplex on a precursor mt-RNA containing the 16S rRNA indicating that mitoribosomal biogenesis proceeds co-transcriptionally. Taken together our data show that RNA processing links transcription to translation via assembly of the mitoribosome. Total RNA was isolated from heart tissue from 11 week old control (Mrpp3loxP/loxP) and Mrpp3 knockout mice (Mrpp3loxP/loxP, +/Ckmm), TruSeq libraries produced in triplicate, sequenced and analysed for differential expression. Mitochondrial RNA was isolated from heart tissue from 11 week old control (Mrpp3loxP/loxP) and Mrpp3 knockout mice (Mrpp3loxP/loxP, +/Ckmm), PARE libraries produced in triplicate and sequenced for analysis of mitochondrial RNA processing.
2016-08-04 | E-GEOD-83471 | biostudies-arrayexpress
Project description:Accurate assembly of newly- synthesized proteins into functional oligomers is crucial for cell activity. In this study, we investigated whether direct interaction of two nascent proteins, emerging from nearby ribosomes (co-co assembly), constitutes a general mechanism for oligomer formation. We used a proteome-wide screen to detect nascent chain-connected ribosome pairs and identified hundreds of homomer subunits that co-co assemble in human cells. Interactions are mediated by five major domain classes, among which N-terminal coiled coils are the most prevalent. We were able to reconstitute co-co assembly of nuclear lamin in Escherichia coli, demonstrating that dimer formation is independent of dedicated assembly machineries. Co-co assembly may thus represent an efficient way to limit protein aggregation risks posed by diffusion-driven assembly routes and ensure isoform-specific homomer formation.
2020-12-18 | GSE151959 | GEO