Bacterial ribosome pause sites surveyed by an integration of ribosome profiling and nascent chain profiling II
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ABSTRACT:
PROVIDER: PRJNA748395 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR15201722.fastq.gz | Fastqsanger.gz | |||
| SRR15201723.fastq.gz | Fastqsanger.gz |
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Project description:Ribosome pauses are associated with diverse co-translational events and determine the fate of mRNAs and proteins. Thus the identification of the precise pause sites across transcriptome is a key, however, the landscape in bacterial has remained ambiguous. Here, we harnessed the multiple ribosome profiling strategies (standard, high-salt-wash, and disome) to survey the robust ribosome pause sites in E. coli. The found pause sites showed the correspondence with biochemical validation by integrated nascent chain profiling (iNP), which detects polypeptidyl-tRNA, an elongation intermediate. Among the list, ribosome pause at Asn586 of ycbZ was ensured by biochemical reporter assay, tRNA-seq, and cryo-electron microscopy. Our results provide a useful resource of ribosome stalling sites in bacteria.
2021-11-23 | GSE160623 | GEO
Project description:Bacterial ribosome pause sites surveyed by an integration of ribosome profiling and nascent chain profiling
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Project description:Ribosome pauses are associated with diverse co-translational events and determine the fate of mRNAs and proteins. Thus the identification of the precise pause sites across transcriptome is a key, however, the landscape in bacterial has remained ambiguous. Here, we harnessed the multiple ribosome profiling strategies (standard, high-salt-wash, and disome) to survey the robust ribosome pause sites in E. coli. The found pause sites showed the correspondence with biochemical validation by integrated nascent chain profiling (iNP), which detects polypeptidyl-tRNA, an elongation intermediate. Among the list, ribosome pause at Asn586 of ycbZ was ensured by biochemical reporter assay, tRNA-seq, and cryo-electron microscopy. Our results provide a useful resource of ribosome stalling sites in bacteria.
2021-11-23 | GSE180482 | GEO
Project description:We use ribosome profiling to demonstrate the selectivity of a small molecule, PF-06446846 that inhibits translation of its target by selectively inducing ribosome-stalling in a nascent chain sequence dependent manner.
2017-06-05 | GSE94454 | GEO
Project description:Transcription by RNA polymerase (RNAP) is interrupted by pauses that play diverse regulatory roles. Although individual pauses have been studied in vitro, the determinants of pauses in vivo and their distribution throughout the bacterial genome remain unknown. Using nascent transcript sequencing we identify a 16 nt consensus pause sequence in E. coli that accounts for known regulatory pause sites as well as ~20,000 new in vivo pause sites. In vitro single-molecule and ensemble analyses demonstrate that these pauses result from RNAP/nucleic-acid interactions that inhibit next-nucleotide addition. The consensus sequence also leads to pausing by RNAPs from diverse lineages and is enriched at translation start sites in both E. coli and B. subtilis. Our results thus implicate a conserved mechanism unifying known and newly identified pause events. Examination of nascent transcripts in E. coli and B. subtilis. 6 samples of E. coli NET-seq, 1 sample of E. coli mRNA-seq, and 1 sample of B. subtilis NET-seq.
2014-05-05 | E-GEOD-56720 | biostudies-arrayexpress
Project description:Translational control is a widespread mode of gene regulation in organisms ranging from bacteria to mammals. Computational models posit that translational control of protein expression during elongation is exerted through a traffic jam of multiple ribosomes at ribosome pause sites on mRNAs. Yet neither the in vivo frequency of ribosome traffic jams nor the contribution of such traffic jams to protein expression has been measured in any organism. Here we show that upon starvation for single amino acids in the bacterium Escherichia coli, ribosome traffic jams are pervasive across the transcriptome, but they occur at only a subset of codons cognate to the limiting amino acid, and their severity is determined by the translation efficiency of mRNAs. Surprisingly, a computational model based on the observed traffic jams at ribosome pause sites is quantitatively inconsistent with measured protein synthesis rates. By comparison, a model incorporating abortion of protein synthesis at ribosome pause sites in addition to ribosome traffic jams predicts protein synthesis rate with higher accuracy. Consistent with the latter model, a significant fraction of the nascent polypeptides at ribosome pause sites is degraded through the activity of the transfer-messenger RNA during amino acid starvation in E. coli. Our work provides a minimal, experimentally-constrained model for predicting protein expression from ribosome dynamics, and it suggests the existence of a trade-off between the cellular translational capacity and the processivity of protein synthesis in vivo. 6 samples for ribosome profiling and 5 samples for total mRNA profiling
2014-05-15 | E-GEOD-51052 | biostudies-arrayexpress
Project description:The yeast Hsp70 chaperone Ssb interacts with ribosomes and nascent chains to co-translationally assist protein folding. Here, we present a proteome-wide analysis of Hsp70 function during translation, based on in vivo selective ribosome profiling, that reveals mechanistic principles coordinating translation with chaperone-assisted protein folding. Ssb binds most cytosolic, nuclear, and mitochondrial proteins and a subset of ER proteins, supporting its general chaperone function. Position-resolved analysis of Ssb engagement reveals compartment- and protein-specific nascent chain binding profiles that are coordinated by emergence of positively charged peptide stretches enriched in aromatic amino acids. Ssbs’ function is temporally coordinated by RAC but independent from NAC. Analysis of ribosome footprint densities along orfs reveals that ribosomes translate faster at times of Ssb binding. This is coordinated by biases in mRNA secondary structure, and codon usage as well as the action of Ssb, suggesting chaperones may allow higher protein synthesis rates by actively coordinating protein synthesis with co-translational folding.
2017-07-17 | GSE93830 | GEO
Project description:The folding of most proteins occurs during the course of their translation while their tRNA-bound C-termini are embedded in the ribosome. How the close proximity of nascent proteins to the ribosome influences their folding thermodynamics remains poorly understood. Here, we have developed a mass spectrometry-based approach for determining the stabilities of nascent polypeptide chains using methionine oxidation as a folding probe. This approach enables quantitative measurements sub-global folding stabilities of ribosome nascent chains (RNCs) within complex protein mixtures and extracts. To validate the methodology, we analyzed the folding thermodynamics of three model proteins (DHFR, CheY and DinB) in soluble and ribosome-bound states. The data indicated that the ribosome can significantly alter the stability of nascent polypeptides. Ribosome-induced stability modulations were highly variable among different folding domains and were dependent on localized charge distributions within nascent polypeptides. The results implicated electrostatic interactions between the ribosome surface and nascent polypeptides as the cause of ribosome-induced stability modulations. The study establishes a robust proteomic methodology for analyzing localized stabilities within ribosome-bound nascent polypeptides and sheds light on how the ribosome influences the thermodynamics of protein folding.
2023-08-09 | PXD037995 | Pride
Project description:Ageing-dependent ribosome pausing and nascent polypeptide aggregation
| PRJNA640641 | ENA
Project description:Ribosome pausing slows down translation and can affect protein synthesis. Improving translation efficiency can therefore be of commercial value. Here, we investigated the occurrence of ribosome pausing during amylase secretion by the industrial production organism Bacillus subtilis under semi fed-batch fermentation conditions. We first assessed our ribosome profiling setup by inducing ribosome stalling at isoleucine codons using the antibiotic mupirocin, and found a pause preference for isoleucine codons preceded by E and P site codons with guanosine residues in their first nucleotide position. Interestingly, when we applied standard ribosomal profiling conditions we found again an enrichment of guanosine residues in the E and P site of ribosome pause sites, but this time also upstream of the ribosome pause site. This sequence motif deviates from previously described ribosome pausing motifs. For the highly expressed amylase gene amyM several strong ribosome pausing sites were detected, which remained present during the 64-hour long fermentation, and were neither related to rare codons nor to secondary protein structures. When surveying the genome, an interesting finding was the presence of strong ribosome pausing sites in several toxins genes. These potential ribosome stall sites may function in preventing inadvertent activity in the cytosol.
2024-01-01 | GSE250313 | GEO