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International Journal of Molecular... May 2024The evolution of the translation system is a fundamental issue in the quest for the origin of life. A feasible evolutionary scenario necessitates the autonomous... (Review)
Review
The evolution of the translation system is a fundamental issue in the quest for the origin of life. A feasible evolutionary scenario necessitates the autonomous emergence of a protoribosome capable of catalyzing the synthesis of the initial peptides. The peptidyl transferase center (PTC) region in the modern ribosomal large subunit is believed to retain a vestige of such a prebiotic non-coded protoribosome, which would have self-assembled from random RNA chains, catalyzed peptide bond formation between arbitrary amino acids, and produced short peptides. Recently, three research groups experimentally demonstrated that several distinct dimeric constructs of protoribosome analogues, derived predicated on the approximate 2-fold rotational symmetry inherent in the PTC region, possess the ability to spontaneously fold, dimerize, and catalyze the formation of peptide bonds and of short peptides. These dimers are examined, aiming at retrieving information concerned with the characteristics of a prebiotic protoribosome. The analysis suggests preconditions for the laboratory re-creation of credible protoribosome analogues, including the preference of a heterodimer protoribosome, contradicting the common belief in the precedence of homodimers. Additionally, it derives a dynamic process which possibly played a role in the spontaneous production of the first bio-catalyzed peptides in the prebiotic world.
Topics: Ribosomes; Peptides; Origin of Life; Peptidyl Transferases; Protein Biosynthesis
PubMed: 38732179
DOI: 10.3390/ijms25094960 -
MSphere May 2024Cervimycins A-D are bis-glycosylated polyketide antibiotics produced by HKI 0179 with bactericidal activity against Gram-positive bacteria. In this study, cervimycin C...
Cervimycins A-D are bis-glycosylated polyketide antibiotics produced by HKI 0179 with bactericidal activity against Gram-positive bacteria. In this study, cervimycin C (CmC) treatment caused a spaghetti-like phenotype in 168, with elongated curved cells, which stayed joined after cell division, and exhibited a chromosome segregation defect, resulting in ghost cells without DNA. Electron microscopy of CmC-treated (3 × MIC) revealed swollen cells, misshapen septa, cell wall thickening, and a rough cell wall surface. Incorporation tests in indicated an effect on DNA biosynthesis at high cervimycin concentrations. Indeed, artificial downregulation of the DNA gyrase subunit B gene () increased the activity of cervimycin in agar diffusion tests, and, in high concentrations (starting at 62.5 × MIC), the antibiotic inhibited DNA gyrase supercoiling activity . To obtain a more global view on the mode of action of CmC, transcriptomics and proteomics of cervimycin treated versus untreated cells were performed. Interestingly, 3 × MIC of cervimycin did not induce characteristic responses, which would indicate disturbance of the DNA gyrase activity . Instead, cervimycin induced the expression of the CtsR/HrcA heat shock operon and the expression of autolysins, exhibiting similarity to the ribosome-targeting antibiotic gentamicin. In summary, we identified the DNA gyrase as a target, but at low concentrations, electron microscopy and omics data revealed a more complex mode of action of cervimycin, which comprised induction of the heat shock response, indicating protein stress in the cell.IMPORTANCEAntibiotic resistance of Gram-positive bacteria is an emerging problem in modern medicine, and new antibiotics with novel modes of action are urgently needed. Secondary metabolites from species are an important source of antibiotics, like the cervimycin complex produced by HKI 0179. The phenotypic response of and toward cervimycin C indicated a chromosome segregation and septum formation defect. This effect was at first attributed to an interaction between cervimycin C and the DNA gyrase. However, omics data of cervimycin treated versus untreated cells indicated a different mode of action, because the stress response did not include the SOS response but resembled the response toward antibiotics that induce mistranslation or premature chain termination and cause protein stress. In summary, these results point toward a possibly novel mechanism that generates protein stress in the cells and subsequently leads to defects in cell and chromosome segregation.
Topics: Anti-Bacterial Agents; Staphylococcus aureus; Microbial Sensitivity Tests; Streptomyces; Bacillus subtilis; Polyketides; Glycosides; Cell Wall; Proteomics; Bacterial Proteins; DNA Gyrase
PubMed: 38722162
DOI: 10.1128/msphere.00764-23 -
BioRxiv : the Preprint Server For... Apr 2024Eukaryotic translation initiation factor (eIF) 3 is a multi-subunit protein complex that binds both ribosomes and messenger RNAs (mRNAs) in order to drive a diverse set...
Eukaryotic translation initiation factor (eIF) 3 is a multi-subunit protein complex that binds both ribosomes and messenger RNAs (mRNAs) in order to drive a diverse set of mechanistic steps during translation. Despite its importance, a unifying framework explaining how eIF3 performs these numerous activities is lacking. Using single-molecule light scattering microscopy, we demonstrate that eIF3 is an equilibrium mixture of the full complex, subcomplexes, and subunits. By extending our microscopy approach to an reconstituted eIF3 and complementing it with biochemical assays, we define the subspecies comprising this equilibrium and show that, rather than being driven by the full complex, mRNA binding by eIF3 is instead driven by the eIF3a subunit within eIF3a-containing subcomplexes. Our findings provide a mechanistic model for the role of eIF3 in the mRNA recruitment step of translation initiation and establish a mechanistic framework for explaining and investigating the other activities of eIF3.
PubMed: 38712078
DOI: 10.1101/2024.04.25.581977 -
Frontiers in Molecular Biosciences 2024Dormant ribosomes are typically associated with preservation factors to protect themselves from degradation under stress conditions. Stm1/SERBP1 is one such protein...
Dormant ribosomes are typically associated with preservation factors to protect themselves from degradation under stress conditions. Stm1/SERBP1 is one such protein that anchors the 40S and 60S subunits together. Several proteins and tRNAs bind to this complex as well, yet the molecular mechanisms remain unclear. Here, we reported the cryo-EM structures of five newly identified Stm1/SERBP1-bound ribosomes. These structures highlighted that eIF5A, eEF2, and tRNA might bind to dormant ribosomes under stress to avoid their own degradation, thus facilitating protein synthesis upon the restoration of growth conditions. In addition, Ribo-seq data analysis reflected the upregulation of nutrient, metabolism, and external-stimulus-related pathways in the strain, suggesting possible regulatory roles of Stm1. The knowledge generated from the present work will facilitate in better understanding the molecular mechanism of dormant ribosomes.
PubMed: 38698775
DOI: 10.3389/fmolb.2024.1395220 -
International Journal of Biological... May 2024Ribosomes, intercellular macromolecules responsible for translation in the cell, are composed of RNAs and proteins. While rRNA makes the scaffold of the ribosome and...
Ribosomes, intercellular macromolecules responsible for translation in the cell, are composed of RNAs and proteins. While rRNA makes the scaffold of the ribosome and directs the catalytic steps of protein synthesis, ribosomal proteins play a role in the assembly of the subunits and are essential for the proper structure and function of the ribosome. To date researchers identified heterogeneous ribosomes in different developmental and growth stages. We hypothesized that under stress conditions the heterogeneity of the ribosomes may provide means to prepare the cells for quick recovery. Therefore the aim of the study was the identification of heterogeneity of ribosomal proteins within the ribosomes in response to eleven stress conditions in Saccharomyces cerevisiae, by means of a liquid chromatography/high resolution mass spectrometry (LC-HRMS) and translation activity tests. Out of the total of 74 distinct ribosomal proteins identified in the study 14 small ribosomal subunit (RPS) and 8 large ribosomal subunit (RPL) proteins displayed statistically significant differential abundances within the ribosomes under stress. Additionally, significant alterations in the ratios of 7 ribosomal paralog proteins were observed. Accordingly, the translational activity of yeast ribosomes was altered after UV exposure, during sugar starvation, cold shock, high salt, anaerobic conditions, and amino acid starvation.
Topics: Saccharomyces cerevisiae; Ribosomal Proteins; Protein Biosynthesis; Ribosomes; Stress, Physiological; Saccharomyces cerevisiae Proteins
PubMed: 38697435
DOI: 10.1016/j.ijbiomac.2024.132004 -
Biochemical Society Transactions Jun 2024Ribosomes are universally conserved cellular machines that catalyze protein biosynthesis. The active sites underly immense evolutionary conservation resulting in... (Review)
Review
Ribosomes are universally conserved cellular machines that catalyze protein biosynthesis. The active sites underly immense evolutionary conservation resulting in virtually identical core structures of ribosomes in all domains of life including organellar ribosomes. However, more peripheral structures of cytosolic ribosomes changed during evolution accommodating new functions and regulatory options. The expansion occurred at the riboprotein level, including more and larger ribosomal proteins and at the RNA level increasing the length of ribosomal RNA. Expansions within the ribosomal RNA occur as clusters at conserved sites that face toward the periphery of the cytosolic ribosome. Recent biochemical and structural work has shed light on how rRNA-specific expansion segments (ESs) recruit factors during translation and how they modulate translation dynamics in the cytosol. Here we focus on recent work on yeast, human and trypanosomal cytosolic ribosomes that explores the role of two specific rRNA ESs within the small and large subunit respectively. While no single regulatory strategy exists, the absence of ESs has consequences for proteomic stability and cellular fitness, rendering them fascinating evolutionary tools for tailored protein biosynthesis.
Topics: RNA, Ribosomal; Humans; Ribosomes; Protein Biosynthesis; Ribosomal Proteins; Nucleic Acid Conformation; Saccharomyces cerevisiae
PubMed: 38695725
DOI: 10.1042/BST20231106 -
ELife Apr 2024The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the...
The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the 'WIN' site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anticancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in human MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anticancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.
Topics: Humans; Antineoplastic Agents; Cell Cycle Proteins; Cell Line, Tumor; Histone-Lysine N-Methyltransferase; Intracellular Signaling Peptides and Proteins; Myeloid-Lymphoid Leukemia Protein; Nuclear Proteins; Proto-Oncogene Proteins; Ribosomes; Tumor Suppressor Protein p53; Peptidomimetics
PubMed: 38682900
DOI: 10.7554/eLife.90683 -
Journal of the American Chemical Society May 2024The ribosome brings 3'-aminoacyl-tRNA and 3'-peptidyl-tRNAs together to enable peptidyl transfer by binding them in two major ways. First, their anticodon loops are...
The ribosome brings 3'-aminoacyl-tRNA and 3'-peptidyl-tRNAs together to enable peptidyl transfer by binding them in two major ways. First, their anticodon loops are bound to mRNA, itself anchored at the ribosomal subunit interface, by contiguous anticodon:codon pairing augmented by interactions with the decoding center of the small ribosomal subunit. Second, their acceptor stems are bound by the peptidyl transferase center, which aligns the 3'-aminoacyl- and 3'-peptidyl-termini for optimal interaction of the nucleophilic amino group and electrophilic ester carbonyl group. Reasoning that intrinsic codon:anticodon binding might have been a major contributor to bringing tRNA 3'-termini into proximity at an early stage of ribosomal peptide synthesis, we wondered if primordial amino acids might have been assigned to those codons that bind the corresponding anticodon loops most tightly. By measuring the binding of anticodon stem loops to short oligonucleotides, we determined that family-box codon:anticodon pairings are typically tighter than split-box codon:anticodon pairings. Furthermore, we find that two family-box anticodon stem loops can tightly bind a pair of contiguous codons simultaneously, whereas two split-box anticodon stem loops cannot. The amino acids assigned to family boxes correspond to those accessible by what has been termed cyanosulfidic chemistry, supporting the contention that these limited amino acids might have been the first used in primordial coded peptide synthesis.
Topics: Anticodon; Amino Acids; Codon; Ribosomes; Binding Sites; Models, Molecular
PubMed: 38676654
DOI: 10.1021/jacs.4c03644 -
Microorganisms Apr 2024Different bacterial species have dramatically different generation times, from 20-30 min in to about two weeks in . The translation machinery in a cell needs to...
Different bacterial species have dramatically different generation times, from 20-30 min in to about two weeks in . The translation machinery in a cell needs to synthesize all proteins for a new cell in each generation. The three subprocesses of translation, i.e., initiation, elongation, and termination, are expected to be under stronger selection pressure to optimize in short-generation bacteria (SGB) such as than in the long-generation . The initiation efficiency depends on the start codon decoded by the initiation tRNA, the optimal Shine-Dalgarno (SD) decoded by the anti-SD (aSD) sequence on small subunit rRNA, and the secondary structure that may embed the initiation signals and prevent them from being decoded. The elongation efficiency depends on the tRNA pool and codon usage. The termination efficiency in bacteria depends mainly on the nature of the stop codon and the nucleotide immediately downstream of the stop codon. By contrasting SGB with long-generation bacteria (LGB), we predict (1) SGB to have more ribosome RNA operons to produce ribosomes, and more tRNA genes for carrying amino acids to ribosomes, (2) SGB to have a higher percentage of genes using AUG as the start codon and UAA as the stop codon than LGB, (3) SGB to exhibit better codon and anticodon adaptation than LGB, and (4) SGB to have a weaker secondary structure near the translation initiation signals than LGB. These differences between SGB and LGB should be more pronounced in highly expressed genes than the rest of the genes. We present empirical evidence in support of these predictions.
PubMed: 38674712
DOI: 10.3390/microorganisms12040768 -
BioRxiv : the Preprint Server For... Apr 2024Co-transcriptional assembly is an integral feature of the formation of RNA-protein complexes that mediate translation. For ribosome synthesis, prior studies have...
Co-transcriptional assembly is an integral feature of the formation of RNA-protein complexes that mediate translation. For ribosome synthesis, prior studies have indicated that the strict order of transcription of rRNA domains may not be obligatory during bacterial ribosome biogenesis, since a series of circularly permuted rRNAs are viable. In this work, we report the insights into assembly of the bacterial ribosome large subunit (LSU) based on cryo-EM density maps of intermediates that accumulate during ribosome synthesis using a set of circularly permuted (CiPer) rRNAs. The observed ensemble of twenty-three resolved ribosome large subunit intermediates reveals conserved assembly routes with an underlying hierarchy among cooperative assembly blocks. There are intricate interdependencies for the formation of key structural rRNA helices revealed from the circular permutation of rRNA. While the order of domain synthesis is not obligatory, the order of domain association does appear to proceed with a particular order, likely due to the strong evolutionary pressure on efficient ribosome synthesis. This work reinforces the robustness of the known assembly hierarchy of the bacterial large ribosomal subunit, and offers a coherent view of how efficient assembly of CiPer rRNAs can be understood in that context.
PubMed: 38644992
DOI: 10.1101/2024.04.10.588894