Ribosome profiling reveals conserved guanosine residues at ribosome pausing sites in Bacillus subtilis
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PROVIDER: PRJNA1053350 | ENA |
REPOSITORIES: ENA
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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
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 | GSE250312 | GEO
Project description:This SuperSeries is composed of the SubSeries listed below.
2024-01-01 | GSE250314 | GEO
Project description:The rate of protein synthesis varies according to the mRNA sequence in ways that affect gene expression. Global analysis of translational pausing is now possible with ribosome profiling. Here, we revisit an earlier report that Shine-Dalgarno sequences are the major determinant of translational pausing in bacteria. Using refinements in the profiling method as well as biochemical assays, we find that SD motifs have little (if any) effect on elongation rates. We argue that earlier evidence of pausing arose from two factors. First, in previous analyses, pauses at Gly codons were difficult to distinguish from pauses at SD motifs. Second, and more importantly, the initial study preferentially isolated long ribosome-protected mRNA fragments that are enriched in SD motifs. These findings clarify the landscape of translational pausing in bacteria as observed by ribosome profiling. Ribosome profiling (three replicates) and RNAseq (two replicates) of E. coli MG1655
2016-01-13 | E-GEOD-72899 | biostudies-arrayexpress
Project description:Limitation for amino acids is thought to regulate translation in mammalian cells primarily by signaling through the kinases mTORC1 and GCN2. We find that limitation for the amino acid arginine causes a selective loss of tRNA charging, which regulates translation through ribosome pausing at two of six arginine codons. Interestingly, limitation for leucine, an essential and abundant amino acid in protein, results in little or no ribosome pausing. Chemical and genetic perturbation of mTORC1 and GCN2 signaling revealed that their robust response to leucine limitation prevents ribosome pausing, while an insufficient response to arginine limitation led to loss of arginine tRNA charging and ribosome pausing. Codon-specific ribosome pausing decreased protein production and triggered premature ribosome termination without significantly reducing mRNA levels. Together, our results suggest that amino acids which are not optimally sensed by the mTORC1 and GCN2 pathways still regulate translation through an evolutionarily conserved mechanism based on synonymous codon usage.
2018-07-19 | GSE113751 | GEO
Project description:Ribosome profiling is a powerful method for globally assessing the activity of ribosomes in a cell. Despite its application in many organisms, ribosome profiling studies in bacteria have struggled to obtain the resolution necessary to precisely define translational pauses. Here we report improvements that yield much higher resolution in E. coli profiling data, enabling us to more accurately assess ribosome pausing and refine earlier studies of the impact of polyproline motifs on elongation. We comprehensively characterize pausing at proline-rich motifs in the absence of elongation factor EFP. We find that only a small fraction of genes with strong pausing motifs have reduced ribosome density downstream and identify features that explain this phenomenon. These features allow us to predict which proteins likely have reduced output in the efp knockout strain. Ribosome profiling of E. coli MG1655 and mutants lacking EFP or its three modifiying enzymes
2015-03-10 | E-GEOD-64488 | biostudies-arrayexpress
Project description:Increased protein misfolding and aggregation are key hallmarks of ageing and many age-related diseases. As generating and maintaining a functional proteome (proteostasis) is crucial to every cellular process, this age-related decline in proteostasis is suggested to play a primary role in the breakdown of diverse cellular subsystems during ageing. Indeed, augmented proteostasis pathways are associated with extended lifespan across different species. With the ribosome as the earliest proteostasis checkpoint, decreased protein synthesis is also a conserved mechanism that extends lifespan. Yet, it remains unclear whether ageing impacts translation after initiation. Here, we use worms and yeast to investigate how ageing influences translation elongation. We show an age-dependent increase in ribosome pausing at diverse positions in coding sequences. This pausing is conserved in both worms and yeast, particularly at Arg and polybasic regions. We further show that such pausing is associated with the age-dependent aggregation of truncated nascent proteins involved in proteostasis pathways, notably aminoacyl tRNA synthetases. Moreover, we find that impairment in clearing truncated nascent proteins is associated with decreased lifespan. These data establish elongation kinetics and co-translational quality control as critical contributors of the age-related proteostasis collapse, which may precipitate the systemic decline observed during ageing.
2021-11-09 | GSE152850 | GEO
Project description:The rate of protein synthesis varies according to the mRNA sequence in ways that affect gene expression. Global analysis of translational pausing is now possible with ribosome profiling. Here, we revisit an earlier report that Shine-Dalgarno sequences are the major determinant of translational pausing in bacteria. Using refinements in the profiling method as well as biochemical assays, we find that SD motifs have little (if any) effect on elongation rates. We argue that earlier evidence of pausing arose from two factors. First, in previous analyses, pauses at Gly codons were difficult to distinguish from pauses at SD motifs. Second, and more importantly, the initial study preferentially isolated long ribosome-protected mRNA fragments that are enriched in SD motifs. These findings clarify the landscape of translational pausing in bacteria as observed by ribosome profiling.
2016-01-13 | GSE72899 | GEO