Translation elongation factor 4 (LepA) contributes to tetracycline susceptibility by stalling elongating ribosomes
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ABSTRACT:
PROVIDER: PRJNA416905 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR6246222.fastq.gz | Fastqsanger.gz | |||
| SRR6246223.fastq.gz | Fastqsanger.gz | |||
| SRR6246224.fastq.gz | Fastqsanger.gz | |||
| SRR6246225.fastq.gz | Fastqsanger.gz | |||
| SRR6246226.fastq.gz | Fastqsanger.gz |
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Project description:Purpose: We use the ribosome profiling protocol to understand EF4 mediated translation events. Methods: We used ribosome profiling data to analyze by plastid software. The ribosome were purified by sucrose gradient separation. Results: Using sequencing data, we found that EF4 mediates the 1-nucleotide conformational change of ribosomes. We also found that many genes involved in translation after tetracycline treatment. Finally, we found that EF4 stalls the elongating ribosomes globally. Conclusions: Our results suggest that EF4 mediates both 30S biogenesis and translation elongation process, in particular, EF4 stalls the elongating ribosomes globally.
2018-05-14 | GSE106448 | GEO
Project description:Even though elongation factor 4 (EF4) is the third most conserved protein in bacteria, its physiological functions remain largely unknown and its proposed molecular mechanisms are conflicting among previous studies. In the present study, we show that the growth of an Escherichia coli strain is more susceptible to tetracycline than its EF4 knockout strain. Consistent with previous studies, our results suggested that EF4 affects ribosome biogenesis when tetracycline is present. Through ribosome profiling analysis, we discovered that EF4 causes 1-nucleotide shifting of ribosomal footprints on mRNA when cells have been exposed to tetracycline. In addition, when tetracycline is present, EF4 inhibits the elongation of protein synthesis, which leads to the accumulation of ribosomes in the early segment of mRNA. Altogether, when cells are exposed to tetracycline, EF4 alters both ribosome biogenesis and the elongation phase of protein synthesis.
| S-EPMC6105825 | biostudies-literature
Project description:In bacteria, translation-transcription coupling inhibits RNA polymerase (RNAP) stalling. We present evidence suggesting that, upon amino acid starvation, inactive ribosomes promote rather than inhibit RNAP stalling. We developed an algorithm to evaluate genome-wide polymerase progression independently of local noise, and used it to reveal that the transcription factor DksA inhibits promoter-proximal pausing and increases RNAP elongation when uncoupled from translation by depletion of charged tRNAs. DksA has minimal effect on RNAP elongation in vitro and on untranslated RNAs in vivo. In these cases, transcripts can form RNA structures that prevent backtracking. Thus, the effect of DksA on transcript elongation may occur primarily upon ribosome slowing/stalling or at promoter-proximal locations that limit the potential for RNA structure. We propose that inactive ribosomes prevent formation of backtrackblocking mRNA structures and that, in this circumstance, DksA acts as a transcription elongation factor in vivo. Chromatin immunoprecipitation (ChIP) experiments were performed by using antibodies against RNA polymerase b subunit in wild-type and DdksA cells treated with 0.5mg/ml serine hydroxamate (SHX) or untreated. DksA and s70 enrichments were compared to RNAP enrichment by ChIP experiments using antibodies against s70 and DksA in wild-type cells (also in DdksA cells as a negative control for DksA ChIP-chip). Differentially labeled ChIP DNA and genomic DNA were competitively hybridized to an E. coli K-12 MG1655 tiling array with overlapping probes at ~12bp spacing across the entire genome. The series contains 19 datasets.
2014-04-21 | E-GEOD-54669 | biostudies-arrayexpress
Project description:In bacteria, translation-transcription coupling inhibits RNA polymerase (RNAP) stalling. We present evidence suggesting that, upon amino acid starvation, inactive ribosomes promote rather than inhibit RNAP stalling. We developed an algorithm to evaluate genome-wide polymerase progression independently of local noise, and used it to reveal that the transcription factor DksA inhibits promoter-proximal pausing and increases RNAP elongation when uncoupled from translation by depletion of charged tRNAs. DksA has minimal effect on RNAP elongation in vitro and on untranslated RNAs in vivo. In these cases, transcripts can form RNA structures that prevent backtracking. Thus, the effect of DksA on transcript elongation may occur primarily upon ribosome slowing/stalling or at promoter-proximal locations that limit the potential for RNA structure. We propose that inactive ribosomes prevent formation of backtrackblocking mRNA structures and that, in this circumstance, DksA acts as a transcription elongation factor in vivo.
2014-04-21 | GSE54669 | GEO
Project description:Stalling of the ribosomes on the mRNA has been linked to myriad of molecular and biological functions. While a multitude of algorithms predict codon-usage in ribosome profiling data, no tool exists to predict the context-specific stalling of ribosomes on transcripts. Combining these two features can reveal unprecedented insights in the ribosomal function and their usage across cells and conditions. To overcome this lack of tools to comprehensively analyze these features, we created Bumpfinder, a web-server, that provides comprehensive analytical details on differences in total ribosome occupancy on transcripts, ribosomal occupancy across codons, ribosome-stalling and the underlying causal amino-acids/codons, as well as collisions. Employing Bumpfinder on generated and publicly available ribosome-profiling datasets, we studied the response of various cell types to amino-acid starvation conditions. We observed interesting patterns in response to the biological effects, suggesting the impact of quality control mechanisms of protein synthesis. Complimentary analysis of collided ribosomes demonstrated phased ribosome stalling on the identified sites. Thus, by deciphering context-specific ribosome distribution, Bumpfinder provides unprecedented molecular insights in the ribosome function and mRNA translation during stress response conditions.
2023-12-31 | GSE156443 | GEO
Project description:Cells can respond to stalled ribosomes by sensing ribosome collisions and employing quality control pathways. How ribosome stalling is resolved without collisions, however, has remained elusive. Here, focusing on non-colliding stalling exhibited by decoding-defective ribosomes, we identified Fap1 as a stalling sensor triggering 18S non-functional rRNA decay via poly-ubiquitination of uS3. Ribosome profiling revealed an enrichment of Fap1 at the translation initiation site but also association with elongating individual ribosomes. Cryo-EM structures of Fap1-bound ribosomes elucidated Fap1 probing the mRNA simultaneously at both the entry and exit channels suggesting a mRNA stasis sensing activity, and Fap1 sterically hinders formation of canonical collided di-ribosomes. Our findings indicate that individual stalled ribosomes are the potential signal for ribosome dysfunction, leading to accelerated turnover of the ribosome itself.
2022-09-15 | GSE159329 | GEO
Project description:Bump-finder: identification of causal codons to the stalling of ribosomes
| PRJNA657892 | ENA
Project description:Problems arising during translation of mRNAs lead to ribosome stalling and collisions with trailing ribosomes. These collisions are known to trigger a series of events including degradation of the stalled nascent polypeptide, decay of the problematic mRNAs, and ribosome rescue. However, the systemic cellular response of ribosome collision has not been explored. Here, we uncover a novel function for ribosome collisions in signal transduction. Using multiple translation elongation inhibitors and cellular stress conditions, we show that ribosome collisions activate both the SAPK (Stress Activated Protein Kinase) and GCN2-mediated cellular stress response pathways that lead to apoptosis and the integrated stress response (ISR), respectively. We further show that the MAPKKK ZAK functions as the sentinel for ribosome collisions, and ZAK is required for activation of both SAPKs p38/JNK and GCN2 signaling pathways. Selective ribosome profiling and biochemistry demonstrate that ZAK preferentially associates with the minimal unit of colliding ribosomes, the disome, inducing ZAK phosphorylation. While ZAK associates with elongating ribosomes under non-stressed conditions, activation of ZAK is specifically trigger by colliding ribosomes. Together, these results provide new insights into how perturbation of translational homeostasis, as read-out by colliding ribosomes, regulates cell fate.
2020-08-18 | GSE141459 | GEO
Project description:Impairment of translation can lead to stalling and collision of ribosomes which constitute an activation platform for several ribosomal stress-surveillance pathways. Among these is the Ribotoxic Stress Response (RSR), where ribosomal sensing by the MAP3K ZAKa leads to activation of p38 and JNK kinases. Despite these insights, the physiological ramifications of ribosomal impairment and downstream RSR signaling remain elusive. Here we show that stalling of ribosomes is sufficient to activate ZAKa. In response to amino acid deprivation and full nutrient starvation, RSR impacts on the ensuing metabolic responses in cells, nematodes and mice. The RSR-regulated responses in these model systems include regulation of AMPK and mTOR signaling, survival under starvation conditions, stress hormone production and regulation of blood sugar control. In addition, ZAK-/- mice present with a lean phenotype. Our work highlights stalled ribosomes as metabolic signals and demonstrates a role for RSR signaling in metabolic regulation.
2022-11-16 | GSE205191 | GEO
Project description:Although several ribosomal protein (RP) paralogs are expressed in a tissue-specific manner, how these proteins affect translation and why they are required only in certain tissues have remained unclear. Here we show that RPL3L, a paralog of RPL3 specifically expressed in heart and skeletal muscle, influences translation elongation dynamics. Deficiency of RPL3L-containing ribosomes (RPL3L-ribosomes) in RPL3L knockout male mice resulted in impaired cardiac contractility. Ribosome occupancy at mRNA codons was found to be altered in the RPL3L-deficient heart, and the changes were negatively correlated with those observed in myoblasts overexpressing RPL3L. RPL3L-ribosomes were less prone to collisions compared with RPL3-containing canonical ribosomes. Although the loss of RPL3L-ribosomes altered translation elongation dynamics for the entire transcriptome, its effects were most pronounced for transcripts related to cardiac muscle contraction and dilated cardiomyopathy, with the abundance of the encoded proteins being correspondingly decreased. Our results provide new insight into the mechanisms and physiological relevance of tissue-specific translational regulation.
2023-03-15 | GSE203072 | GEO