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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 | GSE250312 | 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 | GSE250313 | GEO
Project description:Ribosome profiling reveals conserved guanosine residues at ribosome pausing sites in Bacillus subtilis
| PRJNA1053350 | ENA
Project description:High-throughput methods, such as ribosome profiling, have revealed the complexity of translation regulation in Bacteria and Eukarya with large-scale effects on cellular functions. In contrast, the translational landscape in Archaea remains mostly unexplored. Here, we developed ribosome profiling in a model archaeon, Haloferax volcanii, elucidating, for the first time, the translational landscape of a representative of the third domain of life. We determined the ribosome footprint of H. volcanii to be comparable in size to that of the Eukarya. We linked footprint lengths to initiating and elongating states of the ribosome on leadered transcripts, operons, and on leaderless transcripts, the latter representing 70% of H. volcanii transcriptome. We manipulated ribosome activity with translation inhibitors to reveal ribosome pausing at specific codons. Lastly, we found that the drug harringtonine arrested ribosomes at initiation sites in this archaeon. This drug treatment allowed us to confirm known translation initiation sites and also reveal putative novel initiation sites in intergenic regions and within genes. Ribosome profiling revealed an uncharacterized complexity of translation in this archaeon with bacteria-like, eukarya-like, and potentially novel translation mechanisms. These mechanisms are likely to be functionally essential and to contribute to an expanded proteome with regulatory roles in gene expression.
| S-EPMC7261190 | biostudies-literature
Project description:The journey of a newly synthesized polypeptide starts in the peptidyltransferase center of the ribosome, from where it traverses the exit tunnel. The interior of the ribosome exit tunnel is neither straight nor smooth. How the ribosome dynamics in vivo is influenced by the exit tunnel is poorly understood. Genome-wide ribosome profiling in mammalian cells reveals elevated ribosome density at the start codon and surprisingly the downstream 5th codon position as well. We found that the highly focused ribosomal pausing shortly after initiation is attributed to the geometry of the exit tunnel, as deletion of the loop region from ribosome protein L4 diminishes translational pausing at the 5th codon position. Unexpectedly, the ribosome variant undergoes translational abandonment shortly after initiation, suggesting that there exists an obligatory step between initiation and elongation commitment. We propose that the post-initiation pausing of ribosomes represents an inherent signature of the translation machinery to ensure productive translation.
| S-EPMC4085768 | biostudies-literature
Project description:Under stress conditions, such as nutrient deprivation, bacteria enter into a hibernation stage, which is characterized by the appearance of 100S ribosomal particles. In Escherichia coli, dimerization of 70S ribosomes into 100S requires the action of the ribosome modulation factor (RMF) and the hibernation-promoting factor (HPF). Most other bacteria lack RMF and instead contain a long form HPF (LHPF), which is necessary and sufficient for 100S formation. While some structural information exists as to how RMF and HPF mediate formation of E. coli 100S (Ec100S), structural insight into 100S formation by LHPF has so far been lacking. Here we present a cryo-EM structure of the Bacillus subtilis hibernating 100S (Bs100S), revealing that the C-terminal domain (CTD) of the LHPF occupies a site on the 30S platform distinct from RMF Moreover, unlike RMF, the BsHPF-CTD is directly involved in forming the dimer interface, thereby illustrating the divergent mechanisms by which 100S formation is mediated in the majority of bacteria that contain LHPF, compared to some ?-proteobacteria, such as E. coli.
| S-EPMC5509997 | biostudies-literature
Project description:Ribosomal stalling is used to regulate gene expression and can occur in a species-specific manner. Stalling during translation of the MifM leader peptide regulates expression of the downstream membrane protein biogenesis factor YidC2 (YqjG) in Bacillus subtilis, but not in Escherichia coli. In the absence of structures of Gram-positive bacterial ribosomes, a molecular basis for species-specific stalling has remained unclear. Here we present the structure of a Gram-positive B. subtilis MifM-stalled 70S ribosome at 3.5-3.9 Å, revealing a network of interactions between MifM and the ribosomal tunnel, which stabilize a non-productive conformation of the PTC that prevents aminoacyl-tRNA accommodation and thereby induces translational arrest. Complementary genetic analyses identify a single amino acid within ribosomal protein L22 that dictates the species specificity of the stalling event. Such insights expand our understanding of how the synergism between the ribosome and the nascent chain is utilized to modulate the translatome in a species-specific manner.
| S-EPMC4423224 | biostudies-literature
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.
| S-EPMC4835026 | biostudies-literature
Project description:Members of the third domain of life, the Archaea, are ubiquitous in all environments on Earth but remain understudied in many aspects including regulatory processes of the central dogma. Archaea present unique biology because they utilize a mosaic of molecular features from both Bacteria and Eukarya, along with unique features. The advent of a high-throughput view of the translation landscape via ribosome profiling in the Bacteria and the Eukarya has illuminated the complexity and previously underappreciated regulation of translation (i.e. translation efficiency, ribosome surveillance, etc.) that potentially has large scale effects on cellular functionality. Here, we developed ribosome profiling in a model archaeon, Haloferax volcanii and elucidated the translational landscape for the first time in the third domain of life. We coupled ribosome profiling with translation inhibitors to: (a) determine the size of the archaeal ribosome footprint, (b) systematically assign translation states of the ribosome to footprint lengths in a mostly leaderless transcriptome, (c) experimentally induce ribosome pauses and clarify the pausing landscape comprehensively, (d) identify putative novel proteins, including small open reading frames (smORFs), and (e) provide evidence that many genes initiate on putative alternative translation start sites (paTSS) around and within open reading frames (ORFs), demonstrating how a microorganism with a gene dense genome can produce proteins with distinct functions using the same gene.
2020-04-27 | GSE138990 | GEO
Project description:The ribosome biogenesis GTPase A protein RbgA is involved in the assembly of the large ribosomal subunit in Bacillus subtilis, and homologs of RbgA are implicated in the biogenesis of mitochondrial, chloroplast, and cytoplasmic ribosomes in archaea and eukaryotes. The precise function of how RbgA contributes to ribosome assembly is not understood. Defects in RbgA give rise to a large ribosomal subunit that is immature and migrates at 45 S in sucrose density gradients. Here, we report a detailed biochemical analysis of RbgA and its interaction with the ribosome. We found that RbgA, like most other GTPases, exhibits a very slow k(cat) (14 h(-1)) and has a high K(m) (90 μM). Homology modeling of the RbgA switch I region using the K-loop GTPase MnmE as a template suggested that RbgA requires K(+) ions for GTPase activity, which was confirmed experimentally. Interaction with 50 S subunits, but not 45 S intermediates, increased GTPase activity by ∼55-fold. Stable association with 50 S subunits and 45 S intermediates was nucleotide-dependent, and GDP did not support strong interaction with either of the subunits. GTP and guanosine 5'-(β,γ-imido)triphosphate (GMPPNP) were sufficient to promote association with the 45 S intermediate, whereas only GMPPNP was able to support binding to the 50 S subunit, presumably due to the stimulation of GTP hydrolysis. These results support a model in which RbgA promotes a late step in ribosome biogenesis and that one role of GTP hydrolysis is to stimulate dissociation of RbgA from the ribosome.
| S-EPMC3318688 | biostudies-literature