Translation elongation factor 4 (LepA) contributes to tetracycline susceptibility by stalling elongating ribosomes
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ABSTRACT:
ORGANISM(S): Escherichia coli BW25113
PROVIDER: GSE106448 | GEO | 2018/05/14
REPOSITORIES: GEO
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Project description:Translation elongation factor 4 (LepA) contributes to tetracycline susceptibility by stalling elongating ribosomes
| PRJNA416905 | ENA
Project description:Ribosome profiling — RNase footprinting of ribosome-bound mRNA — has been a unique and powerful method, applied to widespread organisms to survey ribosome traversal along mRNAs. In contrast to eukaryotes, bacterial ribosome footprints show a broad range of sizes, reflecting the differential states of ribosomes. However, the origin remains unclear. Here, we show that rotated state of ribosomes and intramolecular RNA duplexes each extend bacterial ribosome footprints at the 5′ end. Combining elongation inhibitors, cryo-electron microscopy, and ribosome profiling, we demonstrated that the rotated state of ribosomes results in long footprints. Along the subunit rotation, ribosomal protein S1 — a 30S-subunit RNA-binding protein — sterically protects mRNA at the 5′ end of the ribosome from RNase digestion and facilitates elongation of the ribosome. Moreover, we found that ribosomes stalled on ycbZ mRNA generate prolonged footprints because of their unique RNA secondary structure proximal to ribosomes. Through the studies of footprint extension, our results revealed S1-mediated stabilization of translation elongation and provide ribosome profiling approach to probe the conformational diversity of ribosomes in bacteria.
2023-07-28 | GSE155243 | GEO
Project description:Structural biology performed inside cells can capture molecular machines in action within their native context. Here we develop an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae. We combine whole-cell crosslinking mass spectrometry, cellular cryo-electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at sub-nanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpoint NusA at the interface between the ribosome and a NusG-bound elongating RNAP, and propose it could mediate transcription-translation coupling. Transcription inhibition stalls and rearranges the expressome, whereas translation inhibition dissociates it, demonstrating that the elongating expressome architecture requires active translation and transcription within the cell.
2020-07-30 | PXD017695 | Pride
Project description:Structural biology performed inside cells can capture molecular machines in action within their native context. Here we develop an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae. We combine whole-cell crosslinking mass spectrometry, cellular cryo-electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at sub-nanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpoint NusA at the interface between the ribosome and a NusG-bound elongating RNAP, and propose it could mediate transcription-translation coupling. Transcription inhibition stalls and rearranges the expressome, whereas translation inhibition dissociates it, demonstrating that the elongating expressome architecture requires active translation and transcription within the cell.
2020-07-30 | PXD017711 | Pride
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:We use mRNA-seq in combination with polysome profiling to determine translational status for all mRNAs in Drosophila mature oocytes and activated eggs. Puromycin-treated lysates are used as a negative control in polysome profiling experiments. Additionally, we use ribosome footprinting to globally measure translational efficiency of mRNAs in wild type mature oocytes as well as wild type and png mutant activated eggs. Lysates of hand-dissected Drosophila mature oocytes (containing ~540 M-NM-<g of total RNA) were subjected to separation by velocity sedimentation through sucrose gradients. In this way, free mRNAs (present in RNPs fraction) or those comigrating with ribosomal subunits (40S or 60S+80S fractions) or with varying numbers of bound ribosomes (low polysomes (2-4 ribosomes), medium polysomes (5-9 ribosomes), and heavy polysomes (more than 10 ribosomes) can be separated based on their size and collected as sucrose gradient fractions. To compare quantitatively the levels of every mRNA across the polysome gradient fractions, we added 5ng of S. cerevisiae mRNA as an exogenous spike-in to each of the six fractions of interest: RNPs, 40S, 60S+80S, low polysomes, medium polysomes and heavy polysomes. RNA was extraced from these fractions, follwing proteinase K treatment, by hot acid phenol method. In case of unfractionated lysates, RNA was extracted using TRIzol (Invitrogen) according to manufacturerM-bM-^@M-^Ys instructions. mRNA-seq samples were prepared from 1 M-NM-<g of total RNA (in case of sucrose gradient fractions and unfractionated lysates) and subject to Illumina based sequencing. Puromycin-treated lysates of mature oocytes or 0-2h Drosophila activated eggs (containing ~540 M-NM-<g of total RNA) were also subjected to separation by velocity sedimentation through sucrose gradients. Puromycin causes premature termination of elongating ribosomes and thus it can be used to determine whether the mRNAs co-sedimenting with the polysomal peaks (defined here as M-bM-^IM-%5 ribosomes) were actively engaged in translation. As an independent approach to assess translation and obtain information on the position of ribosomes on mRNAs, we employed ribosome footprinting. In addition to analyzing the same samples, as by polysome profiling, we also analyzed png mutant activated eggs by ribosome footprinting. Ribosome footprint profiling measures the number of ribosome-protected fragments (RPFs) derived from the mRNAs of each gene, resulting in a singular value of translational efficiency (TE) for each gene (TE=RPF/RNA).
2014-06-12 | E-GEOD-52799 | biostudies-arrayexpress
Project description:Fully assembled ribosomes exist in two populations: polysomes and monosomes. While the former has been studied extensively, to what extent translation occurs on monosomes and its importance for overall translational output remains controversial. Here, we used ribosome profiling to examine the translational status of 80S monosomes in Saccharomyces cerevisiae. We found that the vast majority of 80S monosomes are elongating, not initiating. Further, most mRNAs exhibit some degree of monosome occupancy, with monosomes predominating on nonsense-mediated decay (NMD) targets, upstream open reading frames (uORFs), canonical ORFs shorter than ~590 nucleotides and ORFs for which the total time required to complete elongation is substantially shorter than that required for initiation. Importantly, mRNAs encoding low-abundance regulatory proteins tend to be enriched in the monosome fraction. Our data highlight the importance of monosomes for the translation of highly regulated mRNAs. We examined the translational status of single 80S ribosomes using ribosome profiling, and compared these monosome footprints to both polysome ribosome footprints and general ribosome profiling. RNASeq libraries were also prepared from the overall sample input.
2016-02-11 | E-GEOD-76117 | biostudies-arrayexpress
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:Ribosome rescue pathways recycle stalled ribosomes and target problematic mRNAs and aborted proteins for degradation. In bacteria, it remains unclear how rescue pathways distinguish ribosomes stalled in the middle of a transcript from actively translating ribosomes. In a genetic screen in E. coli, we discovered a novel rescue factor that has endonuclease activity. SmrB cleaves mRNAs upstream of stalled ribosomes, allowing the ribosome rescue factor tmRNA (which acts on truncated mRNA) to rescue upstream ribosomes. SmrB is recruited by ribosome collisions. Cryo-EM structures of collided disomes from E. coli and B. subtilis reveal interactions between the 30S subunits and a possible SmrB binding site. These findings show that ribosome collisions trigger ribosome rescue in bacteria and reveal the mechanism by which this occurs.
2022-08-01 | GSE179691 | GEO
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