Affinity-purified RqcH-DR-ribosome-associated quality control complexes from Bacillus subtilis
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ABSTRACT:
SUBMITTER: Caillan Thomas Crowe-McAuliffe
PROVIDER: EMPIAR-10726 | biostudies-other |
REPOSITORIES: biostudies-other
Publications
RqcH and RqcP catalyze processive poly-alanine synthesis in a reconstituted ribosome-associated quality control system.
Takada Hiraku H Crowe-McAuliffe Caillan C Polte Christine C Sidorova Zhanna Yu ZY Murina Victoriia V Atkinson Gemma C GC Konevega Andrey L AL Ignatova Zoya Z Wilson Daniel N DN Hauryliuk Vasili V
Nucleic acids research 20210801 14
In the cell, stalled ribosomes are rescued through ribosome-associated protein quality-control (RQC) pathways. After splitting of the stalled ribosome, a C-terminal polyalanine 'tail' is added to the unfinished polypeptide attached to the tRNA on the 50S ribosomal subunit. In Bacillus subtilis, polyalanine tailing is catalyzed by the NEMF family protein RqcH, in cooperation with RqcP. However, the mechanistic details of this process remain unclear. Here we demonstrate that RqcH is responsible fo ...[more]
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| EMPIAR-10540 | biostudies-other
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| EMPIAR-10541 | biostudies-other
Project description:Ribosomes trapped on mRNAs during protein synthesis need to be rescued for the cell to survive. The most ubiquitous bacterial ribosome rescue pathway is trans-translation mediated by tmRNA and SmpB. Genetic inactivation of trans-translation can be lethal, unless ribosomes are rescued by ArfA or ArfB alternative rescue factors or the ribosome-associated quality control (RQC) system, which in Bacillus subtilis involves MutS2, RqcH, RqcP and Pth. Using transposon sequencing in a trans-translation-incompetent B. subtilis strain we identify a poorly characterized S4-domain-containing protein YlmH as a novel potential RQC factor. Cryo-EM structures reveal that YlmH binds peptidyl-tRNA-50S complexes in a position analogous to that of S4-domain-containing protein RqcP, and that, similarly to RqcP, YlmH can co-habit with RqcH. Consistently, we show that YlmH can assume the role of RqcP in RQC by facilitating the addition of poly-alanine tails to truncated nascent polypeptides. While in B. subtilis the function of YlmH is redundant with RqcP, our taxonomic analysis reveals that in multiple bacterial phyla RqcP is absent, while YlmH and RqcH are present, suggesting that in these species YlmH plays a central role in the RQC.
| S-EPMC11317155 | biostudies-literature
Project description:By taking advantage of the strong genetic interactions between trans-translation and other ribosome rescue systems, we have employed a transposon sequencing (Tn-Seq) to identify potential novel rescue factors in Bacillus subtilis. In addition to the identification the ArfA-type rescue factor BrfA, as well as the RQC elongation actors RqcP and RqcH, our Tn-Seq screen led to the identification of YlmH, a poorly characterized S4-domain-containing protein, as a potential RQC factor. Binding of YlmH to 50S ribosomal subunit was confirmed by proteomics approach.
2024-09-16 | PXD049370 | Pride
Project description:Translation arrest by polybasic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control (RQC) system. Here we report that ubiquitination of the 40S ribosomal protein uS10 by the E3 ubiquitin ligase Hel2 (or RQT1) is required for RQC. We identify a RQC-trigger (RQT) subcomplex composed of the RNA helicase-family protein Slh1/Rqt2, the ubiquitin-binding protein Cue3/Rqt3, and yKR023W/Rqt4 that is required for RQC. The defects in RQC of the RQT mutants correlate with sensitivity to anisomycin, which stalls ribosome at the rotated form. Cryo-electron microscopy analysis reveals that Hel2-bound ribosome are dominantly the rotated form with hybrid tRNAs. Ribosome profiling reveals that ribosomes stalled at the rotated state with specific pairs of codons at P-A sites serve as RQC substrates. Rqt1 specifically ubiquitinates these arrested ribosomes to target them to the RQT complex, allowing subsequent RQC reactions including dissociation of the stalled ribosome into subunits.Several protein quality control mechanisms are in place to trigger the rapid degradation of aberrant polypeptides and mRNAs. Here the authors describe a mechanism of ribosome-mediated quality control that involves the ubiquitination of ribosomal proteins by the E3 ubiquitin ligase Hel2/RQT1.
| S-EPMC5534433 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below.
2017-07-31 | GSE90920 | GEO
Project description:Translation elongation stalling has the potential to produce toxic truncated protein fragments. Translation of either non-stop mRNA or transcripts coding for poly-basic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control (RQC) system. During this process, the stalled ribosome is dissociated into subunits, and the polypeptide is ubiquitinated by the E3 ubiquitin ligase Listerin on the 60S large ribosomal subunit, leading to subsequent proteasomal degradation. However, it is largely unknown how the specific stalled ribosomes are recognized as aberrant to engage the RQC system. Here, we report that ubiquitination of the ribosomal protein uS10 of the 40S small ribosomal subunit, by the E3 ubiquitin ligase Hel2 (or RQC-trigger (Rqt) 1) initiates RQC. We identified a novel RQC-trigger (RQT) complex composed of the RNA helicase-family protein Slh1/Rqt2, the ubiquitin binding protein Cue3/Rqt3, and yKR023W/Rqt4 that is required for RQC. The defects in RQC of the RQT mutants correlated with sensitivity to anisomycin, which stalls ribosome at the rotated form, suggesting that RQT factors rescue ribosomes stalled by this drug. Our un-biased survey by ribosome profiling revealed that ribosomes stalled at the rotated state with specific pairs of codons at P-A sites serve as RQC substrates. Rqt1 specifically ubiquitinates these arrested ribosomes to target them to the RQT complex, allowing subsequent RQC reactions including dissociation of the stalled ribosome into subunits. Our results provide mechanistic insight into the surveillance system for aberrant proteins induced by ribosome stalling and mediated by ribosome ubiquitination.
2017-07-31 | GSE90919 | GEO
Project description:Translation elongation rates are regulated to ensure proper conformation and biological function of proteins. Translation of either non-stop mRNA or transcripts coding for poly-basic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control system (RQC). During this process, the stalled ribosome is dissociated into subunits, and the polypeptide is ubiquitinated by the E3 ubiquitin ligase Listerin on the 60S large ribosomal subunit (LSU) leading to subsequent proteasomal degradation. However, it is largely unknown how stalled ribosomes are recognized and dissociated into subunits. Here we report that ubiquitination of the ribosomal protein uS10 by the E3 ubiquitin ligase Hel2 is required for the production of the RQC substrate. RQC-trigger (RQT) factors, a RNA helicase-family protein Slh1/Rqt2, ubiquitin binding protein Cue3/Rqt3 and yKR023W/Rqt4, were also required for the primary steps of RQC, and associated with Hel2-ribosome complexes. Rqt2-4 factors were dispensable for the ubiquitination of uS10 by Hel2/Rqt1 and associated with ribosomes independent of the ubiquitination of uS10. However, the ubiquitin-binding activity of Rqt3 were crucial to trigger RQC. Cryo-electron microscopy (cryo-EM) analysis revealed that Hel2 bound ribosomes are in an rotated state containing hybrid state AP- and PE-tRNAs. Furthermore, ribosome profiling revealed that short footprints, hallmarks of hybrid state ribosomes18, were accumulated at tandem CGA rare codons at the beginning of the poly arginine stalling sequence and long footprints at subsequent codons, respectively. Short footprints at CGA codons were decreased in rqt1 mutant but drastically increased in uS10 mutants defective in the ubiquitination or rqt2 mutant. Collectively, our results demonstrate that Hel2 stabilizes ratcheted ribosomes leading to ubiquitination of uS10. Subsequently, Rqt2-4 factors target these hybrid state ribosomes specifically, allowing subsequent RQC reactions.
2017-07-31 | GSE79622 | GEO
Project description:Potassium and glutamate are the major cation and anion, respectively, in every living cell. Due to the high concentrations of both ions, the cytoplasm of all cells can be regarded as a potassium glutamate solution. This implies that the concentrations of both ions need to be balanced. While the control of potassium uptake by glutamate is well established for eukaryotic cells, much less is known about the mechanisms that link potassium homeostasis to glutamate availability in bacteria. Here, we have discovered that the availability of glutamate strongly decreases the minimal external potassium concentration required for the highly abundant Bacillus subtilis potassium channel KtrCD to accumulate potassium. In contrast, the inducible KtrAB and KimA potassium uptake systems have high apparent affinities for potassium even in the absence of glutamate. Experiments with mutant strains revealed that the KtrD subunit responds to the presence of glutamate. For full activity, KtrD synergistically requires the presence of the regulatory subunit KtrC and of glutamate. The analysis of suppressor mutants of a strain that has KtrCD as the only potassium uptake system and that experiences severe potassium starvation identified a mutation in the ion selectivity filter of KtrD (Gly282 to Val) that similarly results in a strongly glutamate-independent increase of the apparent affinity for potassium. Thus, this work has identified two conditions that increase the apparent affinity of KtrCD for potassium, i.e., external glutamate and the acquisition of a single point mutation in KtrD.IMPORTANCE In each living cell, potassium is required for maintaining the intracellular pH and for the activity of essential enzymes. Like most other bacteria, Bacillus subtilis possesses multiple low- and high-affinity potassium uptake systems. Their activity is regulated by the second messenger cyclic di-AMP. Moreover, the pools of the most abundant ions potassium and glutamate must be balanced. We report two conditions under which the low-affinity potassium channel KtrCD is able to mediate potassium uptake at low external potassium concentrations: physiologically, the presence of glutamate results in a severely increased potassium uptake. Moreover, this is achieved by a mutation affecting the selectivity filter of the KtrD channel. These results highlight the integration between potassium and glutamate homeostasis in bacteria.
| S-EPMC7253608 | biostudies-literature
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.
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