Structure of Escherichia coli heat shock protein Hsp15 in complex with ribosomal 50S sununits bearing peptidyl-tRNA
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PROVIDER: PRJNA892089 | ENA |
REPOSITORIES: ENA
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Project description:In Escherichia coli, the heat shock protein 15 (Hsp15) is part of the cellular response to elevated temperature. Hsp15 interacts with peptidyl-tRNA-50S complexes that arise upon dissociation of translating 70S ribosomes, and is proposed to facilitate their rescue and recycling. A previous structure of E. coli Hsp15 in complex with peptidyl-tRNA-50S complex reported a binding site located at the central protuberance of the 50S subunit. By contrast, recent structures of RqcP, the Hsp15 homolog in Bacillus subtilis, in complex with peptidyl-tRNA-50S complexes have revealed a distinct site positioned between the anticodon-stem-loop (ASL) of the P-site tRNA and H69 of the 23S rRNA. Here we demonstrate that exposure of E. coli cells to heat shock leads to dissociation of 70S ribosomes and accumulation of 50S subunits, thus identifying a natural substrate for Hsp15 binding. Additionally, we have determined a cryo-EM reconstruction of the Hsp15-50S-peptidyl-tRNA complex isolated from heat shocked E. coli cells, revealing that Hsp15 binds to the 50S-peptidyl-tRNA complex analogously to its B. subtilis homolog RqcP. Collectively, our findings support a model where Hsp15 promotes access to the A-site for putative rescue factors to release the aberrant nascent polypeptide chain.
2022-11-15 | GSE216100 | GEO
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2025-04-07 | GSE200478 | GEO
Project description:In Escherichia coli, the heat shock protein 15 (Hsp15) is part of the cellular response to elevated temperature. Hsp15 interacts with peptidyl-tRNA-50S complexes that arise upon dissociation of translating 70S ribosomes, and is proposed to facilitate their rescue and recycling. A previous structure of E. coli Hsp15 in complex with peptidyl-tRNA-50S complex reported a binding site located at the central protuberance of the 50S subunit. By contrast, recent structures of RqcP, the Hsp15 homolog in Bacillus subtilis, in complex with peptidyl-tRNA-50S complexes have revealed a distinct site positioned between the anticodon-stem-loop (ASL) of the P-site tRNA and H69 of the 23S rRNA. Here we demonstrate that exposure of E. coli cells to heat shock leads to a decrease in 70S ribosomes and accumulation of 50S subunits, thus identifying a natural substrate for Hsp15 binding. Additionally, we have determined a cryo-EM reconstruction of the Hsp15-50S-peptidyl-tRNA complex isolated from heat shocked E. coli cells, revealing that Hsp15 binds to the 50S-peptidyl-tRNA complex analogously to its B. subtilis homolog RqcP. Collectively, our findings support a model where Hsp15 stabilizes the peptidyl-tRNA in the P-site and thereby promotes access to the A-site for putative rescue factors to release the aberrant nascent polypeptide chain.
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Project description:Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that prevents the accumulation of harmful truncated proteins by degrading transcripts with premature termination codons (PTCs). NMD activity varies across many contexts, with the variation having critical consequences during both normal development and disease. However, the factors that influence this variability remain poorly understood. Here, we investigate the relationship between the immediate sequence context of a PTC and NMD activity. We find an enrichment of glycine (Gly) codons preceding a PTC in common nonsense variants in contrast with a depletion of Gly codons preceding a normal termination codon (NTC). Furthermore, Gly-PTC enrichment is most pronounced in genes that tolerate loss-of-function variants, suggesting a biological impact for Gly-PTC contexts. Consistent with this idea, Gly-PTC contexts have higher NMD activity compared to an alanine-PTC context. We used a massively parallel reporter assay to comprehensively assess the effect of PTC sequence context on NMD activity, which revealed that peptidyl-tRNA hydrolysis rate during translation termination was the most important feature in discriminating high and low NMD activity. We show with biochemical assays in a eukaryotic system that Gly-TC contexts have the slowest termination rate compared to other codons. Based on these findings, we propose that NMD activity is modulated by the "window of opportunity" offered by peptidyl-tRNA hydrolysis rate and thus, translation termination kinetics. Gly-PTC contexts may have coevolved with NMD to ensure robust elimination of potentially toxic truncated proteins from non-essential genes.
2025-03-10 | GSE261151 | GEO
Project description:Cells adapt to environmental stressors such as heat shock and extracellular acidosis through formation of nuclear membrane-less compartments called Amyloid bodies (A-bodies). Stressors activate formation of Amyloid bodies (A-bodies) via induction of ribosomal intergenic spacer RNA (rIGSRNA). RNA-seq on non-ribosome depleted RNA from human MCF7 cells exposed to heat shock (43C, 30 minutes) revealed the heat shock-specific expression profile of rIGSRNA.
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Project description:Heat shock-induced ribosomal intergenic spacer RNA
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Project description:Peptidyl-tRNA hydrolysis rate influences the efficiency of nonsense-mediated mRNA decay
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Project description:Protein translation is an essential cellular process, but paradoxically, it can also promote aging and other stresses. In a screen for mutations that protect C. elegans from hypoxic stress, we isolated multiple genes that impact translation, including a ribosomal RNA helicase gene, ddx-52, tRNA biosynthesis genes, and a gene controlling amino acid availability. To better define the mechanisms whereby these genes control hypoxic injury, we performed a second screen for genetic suppressors of the ddx-52(lf) phenotypes. This screen identified multiple genes involved in ribosome biogenesis. Surprisingly, the suppressor mutations were able to restore normal hypoxic sensitivity and protein synthesis to the tRNA biosynthesis mutants, but not to the mutant reducing amino acid uptake. Proteomic characterization of the mutants demonstrated that reduced tRNA biosynthetic activity produces a corresponding reduction in ribosomal structural subunits. Our study uncovers an uncharacterized regulatory interaction between ribosomal biogenesis and tRNA abundance controlling translation and hypoxic survival. A proteomic analysis of a threonyl-tRNA synthetase mutant with and without the ribosomal biogenesis suppressors revealed an uncharacterized feedback mechanism whereby disruption of tRNA aminoacylation results in a corresponding reduction in ribosomal subunits, thereby explaining the ability of enhanced ribosomal biogenesis to suppress reduced tRNA aminoacylation.
2022-02-15 | PXD017267 | Pride
Project description:We report the context-specific activity of two peptidyl transferase targeting antibiotics, chloramphenicol and linezolid. By generating ribosome profiling data in the presence or absence of either chloramphenicol or linezolid we mapped the relative change of ribosome density induced by these antibiotics. We find that both inhibitors preferentially arrest ribosomes that carry either an alanine, serine, or threonine in the penultimate (-1) position of the nascent peptide chain. Additionally ribosomes that carry a glycine in either the P site (0) or A-site (+1) counteract the inhibitory activity of both inhibitors. The context-specific action of chloramphenicol illuminates the operation of the mechanism of inducible resistance that relies on programmed drug-induced translation arrest. In addition, our findings expose the functional interplay between the nascent chain and the peptidyl transferase center.
2016-09-08 | GSE86536 | GEO
Project description:These are the ribosomal subunit fractions from the polysome gradients. investigating effect of heat shock on procyclic-form trypanosomes.
2022-01-22 | E-MTAB-11314 | biostudies-arrayexpress