R-proteins in modification deficient ribosomes - dissociated 50S subunits from 70S
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap XL
ORGANISM(S): Escherichia Coli
SUBMITTER: 
Kaspar Reier
LAB HEAD: Tiina Tamm
PROVIDER: PXD047588 | Pride | 2024-03-15
REPOSITORIES: Pride
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Project description:Ribosomal RNA modifications are introduced by specific enzymes during ribosome assembly in bacteria. Deletion of individual modification enzymes has a minor effect on bacterial growth, ribosome biogenesis, and translation, which has complicated the definition of the function of the enzymes and their products. We have constructed an E. coli strain lacking 10 genes encoding enzymes that modify 23S rRNA around the peptidyl-transferase center. This strain exhibits severely compromised growth and ribosome assembly, especially at lower temperatures. Ribosomal protein composition of the mature 50S ribosomes and free 50S subunits was analyzed by SILAC based qMS approach. We have found that absence of 23S rRNA modifications around PTC does not affect significantly r-protein content of incompletely assembled 50S or mature 50S ribosomes.
2024-03-15 | PXD047376 | Pride
Project description:The 120-nt long 5S rRNA, is an indispensable component of cytoplasmic ribosomes in all living organisms. The functions of 5S rRNA and the reasons for its evolutionary preservation as an independent molecule remain unclear. Here we used ribosome engineering to investigate whether maintaining 5S rRNA as an independent molecule is critical for ribosome function and cell survival. By fusing circularly permutated 5S rRNA (cp5S) with 23S rRNA and deleting all wild type 5S rRNA genes, we generated an Escherichia coli strain completely devoid of free 5S rRNA. Viability of the engineered cells demonstrates that autonomous 5S rRNA is not required for cell growth at 37°C and is unlikely to have essential functions outside the ribosome. The fully-assembled ribosomes carrying 23S-cp5S hybrid rRNA and lacking free 5S rRNA are highly active in translation. However, the engineered cells accumulate aberrant 50S subunits that are unable to form stable 70S ribosomes. Cryo-EM analysis revealed a dramatically malformed peptidyl transferase center in the misassembled 50S subunits. The results of our experiments argue that the key evolutionary force preserving the autonomous nature of the smallest rRNA is its role in ribosome biogenesis.
2020-06-18 | PXD019058 | Pride
Project description:23S ribosomal RNA (rRNA) of Escherichia coli 50S large ribosome subunit contains 26 post-transcriptionally modified nucleosides. Here, we determine the extent of modifications in the 35S and 45S large subunit intermediates, accumulating in cells expressing the helicase inactive DbpA protein, R331A, and the native 50S large subunit. The modifications we characterized are nine pseudouridines, 3-methylpseudouridine, 2-methyladenine, and 5-hydroxycytosine. These modifications were detected using 1-cyclohexyl-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate (CMCT) treatment followed by alkaline treatment. In addition, KMnO4 treatment of 23S rRNA was employed to detect 5-hydroxycytosine modification. CMCT and KMnO4 treatment produce chemical changes in modified nucleotides that cause reverse transcriptase misincorporations and deletions, which were detected employing next generation sequencing. Our results show that seven uridines to pseudouridine isomerizations and the 2-methyladenosine modification are present both in the 35S and 45S to similar extents as in the 50S. Hence, the enzymes that perform these modifications, namely RluF, RluB, RlmN, RluA, RluC, and RluA, have already acted in the intermediates. While two uridines to pseudouridines isomerizations, the 3-methylpseudouridine, and 5-hydroxycytosine modifications are significantly less present in the 35S and 45S as compared to the 50S. Therefore, the enzymes that perform these modifications, RluD, RlmH, and RlhA, are in the process of modifying the 35S, and 45S or these enzymes act during the later stages of ribosome assembly. Our study employs a novel high throughput and single nucleotide resolution technique for detection of 2-methyladenine and two novel high throughput and single nucleotide resolution technique for detection of 5-hydroxycytosine.
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Project description:Ribosomes, macromolecular machines producing a cell´s protein content, are formed from their RNA and protein components in a dynamic process referred to as ribosome assembly. While ribosome assembly in the cell starts with the successive synthesis of ribosomal RNA (rRNA) by the RNA polymerase holoenzyme, and requires numerous assembly factors, (Kaczanowska & Ryden-Aulin, 2007; Shajani et al., 2011), the process can be accomplished in vitro, using purified ribosomal components and scalable reaction conditions (Nierhaus & Dohme, 1974; Traub & Nomura, 1968). To obtain structural and conceptual insights in the early phase of the process, we performed the in vitro assembly reaction of the bacterial 50S subunit as a time course reaction, analyzed samples by sucrose density gradient ultracentrifugation, activity assay, quantitative mass spectrometry (qMS) and cryo-electron microscopy (cryo-EM). Our structural analysis reveals that early 50 assembly initiates with 23S rRNA domain I and occurs in a domain-wise fashion, while late 50S assembly proceeds incrementally. Notably, in both phases parallel pathways are utilized. Furthermore, we find that both ribosomal proteins and folded rRNA helices, occupying surface exposed regions on pre-50S particles, induce, or stabilize rRNA folds within adjacent regions, thereby creating cooperativity. Our study provides new insights into fundamental principles governing ribosome assembly.
2022-09-05 | PXD030312 | Pride
Project description:Biogenesis of plastid ribosomes is facilitated by auxiliary factors which process and modify ribosomal RNAs (rRNA) or are involved in ribosome assembly. In comparison to their bacterial and mitochondrial counterparts, the biogenesis of plastid ribosomes is less well understood and few auxiliary factors have been described so far. In this study, we report the functional characterization of CGL20 (CONSERVED ONLY IN THE GREEN LINEAGE20) in Arabidopsis thaliana (AtCGL20) a small, proline-rich protein which is targeted to mitochondria and chloroplasts. In Arabidopsis, CGL20 is encoded by segmentally duplicated genes of high similarity (AtCGL20A and AtCGL20B), and inactivation of both in the atcgl20ab mutant leads to a visible virescent phenotype, and growth arrest at low temperature. The chloroplast proteome, thylakoid complex abundance and pigment composition are significantly affected in atcgl20ab mutants, resulting in a combined proton gradient regulation (pgr) and chlororespiratory reduction (crr) phenotype. Loss of AtCGL20 alters plastid rRNA ratios, perturbs the formation of a hidden break in 23S rRNA and causes abnormal accumulation of 50S ribosomal subunits in the high-molecular-mass fraction of chloroplast stromal extracts. Moreover, AtCGL20A-eGFP fusion proteins comigrate with 50S ribosomal subunits in sucrose density gradients, even after RNase treatment of stromal extracts. Therefore, we propose that AtCGL20 participates in late stages of the biogenesis of 50S ribosomal subunits in plastids – a role which presumably evolved in the green lineage as a consequence of structural divergence of plastid ribosomes.
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Project description:In Escherichia coli, the heat shock protein 15 (Hsp15) is part of the cellular response to elevated temperature. Hsp15 interacts with peptidyl-tRNA-50S complexes that arise upon dissociation of translating 70S ribosomes, and is proposed to facilitate their rescue and recycling. A previous structure of E. coli Hsp15 in complex with peptidyl-tRNA-50S complex reported a binding site located at the central protuberance of the 50S subunit. By contrast, recent structures of RqcP, the Hsp15 homolog in Bacillus subtilis, in complex with peptidyl-tRNA-50S complexes have revealed a distinct site positioned between the anticodon-stem-loop (ASL) of the P-site tRNA and H69 of the 23S rRNA. Here we demonstrate that exposure of E. coli cells to heat shock leads to dissociation of 70S ribosomes and accumulation of 50S subunits, thus identifying a natural substrate for Hsp15 binding. Additionally, we have determined a cryo-EM reconstruction of the Hsp15-50S-peptidyl-tRNA complex isolated from heat shocked E. coli cells, revealing that Hsp15 binds to the 50S-peptidyl-tRNA complex analogously to its B. subtilis homolog RqcP. Collectively, our findings support a model where Hsp15 promotes access to the A-site for putative rescue factors to release the aberrant nascent polypeptide chain.
2022-11-15 | GSE216100 | GEO
Project description:Lincomycin is a lincosamide antibiotic that forms cross-links within the peptidyl transferase loop region of the 23S rRNA of the 50S subunit of the bacterial ribosome, thereby inhibiting protein synthesis. We have previously reported that lincomycin at concentrations below the minimum inhibitory concentration potentiates the production of secondary metabolites in actinomycete strains. We aimed to elucidate the fundamental mechanisms underlying lincomycin induction of secondary metabolism in actinomycetes. Therefore, the dose-dependent response of lincomycin on gene expression of the model actinomycetes Streptomyces coelicolor A3(2) and possible relationships to secondary metabolism have been investigated.
2018-03-02 | E-MTAB-6434 | biostudies-arrayexpress
Project description:The ribosome is one of the largest and the most complicated enzymes in cells, making up 25% of the bacterial cell’s dry mass, and is responsible for protein synthesis in all organisms on earth. In 2013, Jewett et al. reported an in vitro system named integrated rRNA synthesis, ribosome assembly, and translation (iSAT) reaction, which provided a defined system for ribosome biogenesis in a near-physiological environment.In this project, the r-protein composition in 30S and 50S ribosomeal subunits assembled in the iSAT reaction was quantified by mass spectrometry compared to 15N labeled 70S ribosomes.
2023-05-10 | PXD038716 | Pride
Project description:RNase E, an essential endoribonuclease in Escherichia coli, is the key component of the multienzyme RNA degradosome, which is localized to the inner cytoplasmic membrane. Until now, the reason for membrane localization of RNase E was not known. We have analyzed ribosome assembly in the rneΔMTS strain, which expresses a cytoplasmic variant of RNase E (cRNase E) resulting in a cytoplasmic RNA degradosome. In this mutant strain, there is a mild slowdown in the rates of growth and mRNA degradation. Here we document the striking accumulation of intermediates in ribosome assembly in the rneΔMTS strain in which precursors of 16S and 23S rRNA are cleaved by cRNase E. In vitro, we show that ribosomes partially unfolded in low ionic strength buffer are cleaved by RNase E. Mapping of in vivo and in vitro cleavage sites shows that they overlap and that their consensus sequence matches previously mapped RNase E cleavage sites. In vivo, fragments of 16S and 23S rRNA as well as a precursor of 5S rRNA are degraded in a pathway involving polyadenylation and 3’ exonucleases. Since the pathway for rRNA degradation is the same as the pathway for mRNA degradation, the slowdown of mRNA degradation in the rneΔMTS strain could be due to competition by rRNA degradation. Our results strongly suggest that the RNA degradosome participates in the quality control of ribosome assembly and that localization on the inner cytoplasmic membrane protects newly synthesized intermediates from wasteful degradation. Ribosome synthesis is costly. Since growth rate correlates with ribosome synthesis rate, slow growth rate of the rneΔMTS strain is due to the degradation of a proportion of newly synthesized ribosomes. Avoiding wasteful degradation of intermediates in ribosome assembly likely explains why localization of RNase E homologues to the inner cytoplasmic membrane is conserved throughout the γ-Proteobacteria.
2023-01-16 | PXD038317 | Pride
Project description:In the cell, stalled ribosomes are rescued through ribosome-associated protein quality-control (RQC) pathways. We demonstrate that RqcH is responsible for tRNAAla selection during RQC elongation, whereas RqcP lacks any tRNA specificity. The ribosomal protein uL11 is crucial for RqcH, but not RqcP, recruitment to the 50S subunit, and B. subtilis lacking uL11 are RQC-deficient. Through mutational mapping, we identify critical residues within RqcH and RqcP that are important for interaction with the P-site tRNA and/or the 50S subunit. Collectively, our findings provide mechanistic insight into the role of RqcH and RqcP in the bacterial RQC pathway. the synthesis of C-terminal poly-Alanine ‘tail’. While it is well-established that RqcP and RqcH are essential for poly-Alanine-tailing in living cells, here, by reconstituting the RQC elongation biochemically we provide a direct proof that the two factors are sufficient. We demonstrate that while RqcH is responsible for tRNAAla selection during RQC elongation, RqcP lacks the tRNA specificity. Through mutational mapping we show that stable interaction of RqcP with 23S rRNA – but not with tRNA – is crucial for RQC, supporting the role of the E-site bound RqcP as a pawl of RQC elongation ratchet. We show that ribosomal protein uL11 is crucial for RqcH – but nor RqcP – recruitment to 50S, and B. subtilis lacking uL11 is RQC-deficient. Finally, we show that proteins encoded by non-stop mRNAs are not universally efficiently targeted by RQC, and that simultaneous disruption of RQC and trans-translation results in bacterial filamentation
2021-08-19 | GSE174254 | GEO