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Genetics Dec 2019Ribosomes of Archaea and Eukarya share higher homology with each other than with bacterial ribosomes. For example, there is a set of 35 r-proteins that are specific only...
Ribosomes of Archaea and Eukarya share higher homology with each other than with bacterial ribosomes. For example, there is a set of 35 r-proteins that are specific only for archaeal and eukaryotic ribosomes. Three of these proteins-eL19, eL24, and eL41-participate in interactions between ribosomal subunits. The eukaryote-specific extensions of r-proteins eL19 and eL24 form two intersubunit bridges eB12 and eB13, which are present only in eukaryotic ribosomes. The third r-protein, eL41, forms bridge eB14. Notably, eL41 is found in all eukaryotes but only in some Archaea. It has been shown that bridges eB12 and eB13 are needed for efficient translation, while r-protein eL41 plays a minor role in ribosome function. Here, the functional interactions between intersubunit bridges were studied using budding yeast strains lacking different combinations of the abovementioned bridges/proteins. The growth phenotypes, levels of translation, ribosome-polysome profiles, and association of ribosomal subunits were analyzed. The results show a genetic interaction between r-protein eL41 and the eB12 bridge-forming region of eL19, and between r-proteins eL41 and eL24. It was possible to construct viable yeast strains with Archaea-like ribosomes lacking two or three eukaryote-specific bridges. These strains display slow growth and a poor translation phenotype. In addition, bridges eB12 and eB13 appear to cooperate during ribosome subunit association. These results indicate that nonessential structural elements of r-proteins become highly important in the context of disturbed subunit interactions. Therefore, eukaryote-specific bridges may contribute to the evolutionary success of eukaryotic translation machinery.
Topics: Models, Molecular; Mutation; Phenotype; Polyribosomes; Protein Binding; Protein Domains; Ribosome Subunits; Ribosomes; Saccharomyces cerevisiae
PubMed: 31649153
DOI: 10.1534/genetics.119.302777 -
Molecular Cell Jan 2021In all branches of life, stalled translation intermediates are recognized and processed by ribosome-associated quality control (RQC) pathways. RQC begins with the...
In all branches of life, stalled translation intermediates are recognized and processed by ribosome-associated quality control (RQC) pathways. RQC begins with the splitting of stalled ribosomes, leaving an unfinished polypeptide still attached to the large subunit. Ancient and conserved NEMF family RQC proteins target these incomplete proteins for degradation by the addition of C-terminal "tails." How such tailing can occur without the regular suite of translational components is, however, unclear. Using single-particle cryo-electron microscopy (EM) of native complexes, we show that C-terminal tailing in Bacillus subtilis is mediated by NEMF protein RqcH in concert with RqcP, an Hsp15 family protein. Our structures reveal how these factors mediate tRNA movement across the ribosomal 50S subunit to synthesize polypeptides in the absence of mRNA or the small subunit.
Topics: Bacillus subtilis; Bacterial Proteins; Cryoelectron Microscopy; Ribosome Subunits, Large, Bacterial
PubMed: 33259810
DOI: 10.1016/j.molcel.2020.11.002 -
Nucleic Acids Research Nov 2023Large ribosomal subunit precursors (pre-LSUs) are primarily synthesized in the nucleolus. At an undetermined step in their assembly, they are released into the...
Yeast ribosome biogenesis factors Puf6 and Nog2 and ribosomal proteins uL2 and eL43 act in concert to facilitate the release of nascent large ribosomal subunits from the nucleolus.
Large ribosomal subunit precursors (pre-LSUs) are primarily synthesized in the nucleolus. At an undetermined step in their assembly, they are released into the nucleoplasm. Structural models of yeast pre-LSUs at various stages of assembly have been collected using cryo-EM. However, which cryo-EM model is closest to the final nucleolar intermediate of the LSU has yet to be determined. To elucidate the mechanisms of the release of pre-LSUs from the nucleolus, we assayed effects of depleting or knocking out two yeast ribosome biogenesis factors (RiBi factors), Puf6 and Nog2, and two ribosomal proteins, uL2 and eL43. These proteins function during or stabilize onto pre-LSUs between the late nucleolar stages to early nucleoplasmic stages of ribosome biogenesis. By characterizing the phenotype of these four mutants, we determined that a particle that is intermediate between the cryo-EM model State NE1 and State NE2 likely represents the final nucleolar assembly intermediate of the LSU. We conclude that the release of the RiBi factors Nip7, Nop2 and Spb1 and the subsequent stabilization of rRNA domains IV and V may be key triggers for the release of pre-LSUs from the nucleolus.
Topics: Ribosomal Proteins; Ribosome Subunits, Large; Ribosome Subunits, Large, Eukaryotic; RNA, Ribosomal; RNA-Binding Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 37811893
DOI: 10.1093/nar/gkad794 -
The Journal of Cell Biology Dec 2018The correct assembly of ribosomes from ribosomal RNAs (rRNAs) and ribosomal proteins (RPs) is critical, as indicated by the diseases caused by RP haploinsufficiency and...
The correct assembly of ribosomes from ribosomal RNAs (rRNAs) and ribosomal proteins (RPs) is critical, as indicated by the diseases caused by RP haploinsufficiency and loss of RP stoichiometry in cancer cells. Nevertheless, how assembly of each RP is ensured remains poorly understood. We use yeast genetics, biochemistry, and structure probing to show that the assembly factor Ltv1 facilitates the incorporation of Rps3, Rps10, and Asc1/RACK1 into the small ribosomal subunit head. Ribosomes from Ltv1-deficient yeast have substoichiometric amounts of Rps10 and Asc1 and show defects in translational fidelity and ribosome-mediated RNA quality control. These defects provide a growth advantage under some conditions but sensitize the cells to oxidative stress. Intriguingly, relative to glioma cell lines, breast cancer cells have reduced levels of LTV1 and produce ribosomes lacking RPS3, RPS10, and RACK1. These data describe a mechanism to ensure RP assembly and demonstrate how cancer cells circumvent this mechanism to generate diverse ribosome populations that can promote survival under stress.
Topics: Adaptor Proteins, Signal Transducing; Breast Neoplasms; Cell Line, Tumor; Female; GTP-Binding Proteins; Humans; Neoplasm Proteins; Ribosomal Proteins; Ribosome Subunits, Small, Eukaryotic; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 30348748
DOI: 10.1083/jcb.201804163 -
MBio Dec 2021During nutrient limitation, bacteria produce the alarmones (p)ppGpp as effectors of a stress signaling network termed the stringent response. RsgA, RbgA, Era, and HflX...
During nutrient limitation, bacteria produce the alarmones (p)ppGpp as effectors of a stress signaling network termed the stringent response. RsgA, RbgA, Era, and HflX are four ribosome-associated GTPases (RA-GTPases) that bind to (p)ppGpp in Staphylococcus aureus. These enzymes are cofactors in ribosome assembly, where they cycle between the ON (GTP-bound) and OFF (GDP-bound) ribosome-associated states. Entry into the OFF state occurs upon hydrolysis of GTP, with GTPase activity increasing substantially upon ribosome association. When bound to (p)ppGpp, GTPase activity is inhibited, reducing 70S ribosome assembly and growth. Here, we determine how (p)ppGpp impacts RA-GTPase-ribosome interactions. We show that RA-GTPases preferentially bind to 5'-diphosphate-containing nucleotides GDP and ppGpp over GTP, which is likely exploited as a regulatory mechanism within the cell to shut down ribosome biogenesis during stress. Stopped-flow fluorescence and association assays reveal that when bound to (p)ppGpp, the association of RA-GTPases to ribosomal subunits is destabilized, both and within bacterial cells. Consistently, structural analysis of the ppGpp-bound RA-GTPase RsgA reveals an OFF-state conformation similar to the GDP-bound state, with the G2/switch I loop adopting a conformation incompatible with ribosome association. Altogether, we highlight (p)ppGpp-mediated inhibition of RA-GTPases as a major mechanism of stringent response-mediated ribosome assembly and growth control. The stringent response is a bacterial signaling network that utilizes the nucleotides pppGpp and ppGpp to reprogram cells in order to survive nutritional stresses. However, much about how these important nucleotides control cellular reprogramming is unknown. Our previous work revealed that (p)ppGpp can bind to and inhibit the enzymatic activity of four ribosome-associated GTPases (RA-GTPases), enzymes that facilitate maturation of the 50S and 30S ribosomal subunits. Here, we examine how this occurs mechanistically and demonstrate that this interaction prevents the accommodation of RA-GTPases on ribosomal subunits both and within bacterial cells, with the ppGpp-bound state structurally mimicking the inactive GDP-bound conformation of the enzyme. We additionally reveal that these GTPase enzymes have a greater affinity for OFF-state-inducing nucleotides, which is a mechanism likely to control ribosome assembly during growth. With this, we further our understanding of how ribosome function is controlled by (p)ppGpp, enabling bacterial survival during stress.
Topics: Bacterial Proteins; GTP Phosphohydrolases; Guanosine Pentaphosphate; Guanosine Tetraphosphate; Models, Molecular; Protein Binding; Ribosome Subunits; Staphylococcus aureus
PubMed: 34749534
DOI: 10.1128/mBio.02679-21 -
Cell Reports Apr 2022Synthesis of ribosomes begins in the nucleolus with formation of the 90S pre-ribosome, during which the pre-40S and pre-60S pathways diverge by pre-rRNA cleavage....
Synthesis of ribosomes begins in the nucleolus with formation of the 90S pre-ribosome, during which the pre-40S and pre-60S pathways diverge by pre-rRNA cleavage. However, it remains unclear how, after this uncoupling, the earliest pre-60S subunit continues to develop. Here, we reveal a large-subunit intermediate at the beginning of its construction when still linked to the 90S, the precursor to the 40S subunit. This primordial pre-60S is characterized by the SPOUT domain methyltransferase Upa1-Upa2, large α-solenoid scaffolds, Mak5, one of several RNA helicases, and two small nucleolar RNA (snoRNAs), C/D box snR190 and H/ACA box snR37. The emerging pre-60S does not efficiently disconnect from the 90S pre-ribosome in a dominant mak5 helicase mutant, allowing a 70-nm 90S-pre-60S bipartite particle to be visualized by electron microscopy. Our study provides insight into the assembly pathway when the still-connected nascent 40S and 60S subunits are beginning to separate.
Topics: DEAD-box RNA Helicases; RNA Precursors; RNA, Ribosomal; RNA, Small Nucleolar; Ribosome Subunits, Large, Eukaryotic; Ribosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 35385737
DOI: 10.1016/j.celrep.2022.110640 -
Nucleic Acids Research Apr 2023Understanding the assembly principles of biological macromolecular complexes remains a significant challenge, due to the complexity of the systems and the difficulties...
Understanding the assembly principles of biological macromolecular complexes remains a significant challenge, due to the complexity of the systems and the difficulties in developing experimental approaches. As a ribonucleoprotein complex, the ribosome serves as a model system for the profiling of macromolecular complex assembly. In this work, we report an ensemble of large ribosomal subunit intermediate structures that accumulate during synthesis in a near-physiological and co-transcriptional in vitro reconstitution system. Thirteen pre-50S intermediate maps covering the entire assembly process were resolved using cryo-EM single-particle analysis and heterogeneous subclassification. Segmentation of the set of density maps reveals that the 50S ribosome intermediates assemble based on fourteen cooperative assembly blocks, including the smallest assembly core reported to date, which is composed of a 600-nucleotide-long folded rRNA and three ribosomal proteins. The cooperative blocks assemble onto the assembly core following defined dependencies, revealing the parallel pathways at both early and late assembly stages of the 50S subunit.
Topics: Ribosomes; RNA, Ribosomal; Ribosomal Proteins; Ribosome Subunits, Large
PubMed: 36864669
DOI: 10.1093/nar/gkad082 -
PloS One 2021In yeast and human cells many of the ribosomal proteins (r-proteins) are required for the stabilisation and productive processing of rRNA precursors. Functional coupling...
Analysis of subunit folding contribution of three yeast large ribosomal subunit proteins required for stabilisation and processing of intermediate nuclear rRNA precursors.
In yeast and human cells many of the ribosomal proteins (r-proteins) are required for the stabilisation and productive processing of rRNA precursors. Functional coupling of r-protein assembly with the stabilisation and maturation of subunit precursors potentially promotes the production of ribosomes with defined composition. To further decipher mechanisms of such an intrinsic quality control pathway we analysed here the contribution of three yeast large ribosomal subunit r-proteins rpL2 (uL2), rpL25 (uL23) and rpL34 (eL34) for intermediate nuclear subunit folding steps. Structure models obtained from single particle cryo-electron microscopy analyses provided evidence for specific and hierarchic effects on the stable positioning and remodelling of large ribosomal subunit domains. Based on these structural and previous biochemical data we discuss possible mechanisms of r-protein dependent hierarchic domain arrangement and the resulting impact on the stability of misassembled subunits.
Topics: Fungal Proteins; Protein Folding; RNA Precursors; Ribosomal Proteins; Ribosome Subunits, Large; Saccharomyces cerevisiae
PubMed: 34813592
DOI: 10.1371/journal.pone.0252497 -
Molecular Microbiology Mar 2016Mitochondrial ribosomes of Trypanosoma brucei are composed of 9S and 12S rRNAs, eubacterial-type ribosomal proteins, polypeptides lacking discernible motifs and...
Mitochondrial ribosomes of Trypanosoma brucei are composed of 9S and 12S rRNAs, eubacterial-type ribosomal proteins, polypeptides lacking discernible motifs and approximately 20 pentatricopeptide repeat (PPR) RNA binding proteins. Several PPRs also populate the polyadenylation complex; among these, KPAF1 and KPAF2 function as general mRNA 3' adenylation/uridylation factors. The A/U-tail enables mRNA binding to the small ribosomal subunit and is essential for translation. The presence of A/U-tail also correlates with requirement for translation of certain mRNAs in mammalian and insect parasite stages. Here, we inquired whether additional PPRs activate translation of individual mRNAs. Proteomic analysis identified KRIPP1 and KRIPP8 as components of the small ribosomal subunit in mammalian and insect forms, but also revealed their association with the polyadenylation complex in the latter. RNAi knockdowns demonstrated essential functions of KRIPP1 and KRIPP8 in the actively respiring insect stage, but not in the mammalian stage. In the KRIPP1 knockdown, A/U-tailed mRNA encoding cytochrome c oxidase subunit 1 declined concomitantly with the de novo synthesis of this subunit whereas polyadenylation and translation of cyb mRNA were unaffected. In contrast, the KRIPP8 knockdown inhibited A/U-tailing and translation of both CO1 and cyb mRNAs. Our findings indicate that ribosome-associated PPRs may selectively activate mRNAs for translation.
Topics: Activating Transcription Factors; Animals; Mitochondria; Mitochondrial Proteins; Polyadenylation; Protein Biosynthesis; Proteomics; Protozoan Proteins; RNA Interference; RNA, Messenger; RNA, Protozoan; RNA, Ribosomal; RNA-Binding Proteins; Ribosomal Proteins; Ribosome Subunits, Small; Ribosomes; Trypanosoma brucei brucei
PubMed: 26713541
DOI: 10.1111/mmi.13287 -
Nature Jun 2019The initiation of bacterial translation involves the tightly regulated joining of the 50S ribosomal subunit to an initiator transfer RNA (fMet-tRNA)-containing 30S...
The initiation of bacterial translation involves the tightly regulated joining of the 50S ribosomal subunit to an initiator transfer RNA (fMet-tRNA)-containing 30S ribosomal initiation complex to form a 70S initiation complex, which subsequently matures into a 70S elongation-competent complex. Rapid and accurate formation of the 70S initiation complex is promoted by initiation factors, which must dissociate from the 30S initiation complex before the resulting 70S elongation-competent complex can begin the elongation of translation. Although comparisons of the structures of the 30S and 70S initiation complexes have revealed that the ribosome, initiation factors and fMet-tRNA can acquire different conformations in these complexes, the timing of conformational changes during formation of the 70S initiation complex, the structures of any intermediates formed during these rearrangements, and the contributions that these dynamics might make to the mechanism and regulation of initiation remain unknown. Moreover, the absence of a structure of the 70S elongation-competent complex formed via an initiation-factor-catalysed reaction has precluded an understanding of the rearrangements to the ribosome, initiation factors and fMet-tRNA that occur during maturation of a 70S initiation complex into a 70S elongation-competent complex. Here, using time-resolved cryogenic electron microscopy, we report the near-atomic-resolution view of how a time-ordered series of conformational changes drive and regulate subunit joining, initiation factor dissociation and fMet-tRNA positioning during formation of the 70S elongation-competent complex. Our results demonstrate the power of time-resolved cryogenic electron microscopy to determine how a time-ordered series of conformational changes contribute to the mechanism and regulation of one of the most fundamental processes in biology.
Topics: Cryoelectron Microscopy; Escherichia coli; Peptide Chain Elongation, Translational; Peptide Chain Initiation, Translational; Protein Conformation; Ribosome Subunits, Large, Bacterial; Ribosome Subunits, Small, Bacterial; Ribosomes; Time Factors
PubMed: 31108498
DOI: 10.1038/s41586-019-1249-5