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1.
Philos Trans R Soc Lond B Biol Sci ; 378(1871): 20220038, 2023 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-36633283

RESUMO

Ribosomal incorporation of d-α-amino acids (dAA) and N-methyl-l-α-amino acids (MeAA) with negatively charged sidechains, such as d-Asp, d-Glu, MeAsp and MeGlu, into nascent peptides is far more inefficient compared to those with neutral or positively charged ones. This is because of low binding affinity of their aminoacyl-transfer RNA (tRNA) to elongation factor-thermo unstable (EF-Tu), a translation factor responsible for accommodation of aminoacyl-tRNA onto ribosome. It is well known that EF-Tu binds to two parts of aminoacyl-tRNA, the amino acid moiety and the T-stem; however, the amino acid binding pocket of EF-Tu bearing Glu and Asp causes electric repulsion against the negatively charged amino acid charged on tRNA. To circumvent this issue, here we adopted two strategies: (i) use of an EF-Tu variant, called EF-Sep, in which the Glu216 and Asp217 residues in EF-Tu are substituted with Asn216 and Gly217, respectively; and (ii) reinforcement of the T-stem affinity using an artificially developed chimeric tRNA, tRNAPro1E2, whose T-stem is derived from Escherichia coli tRNAGlu that has high affinity to EF-Tu. Consequently, we could successfully enhance the incorporation efficiencies of d-Asp, d-Glu, MeAsp and MeGlu and demonstrated for the first time, to our knowledge, ribosomal synthesis of macrocyclic peptides containing multiple d-Asp or MeAsp. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.


Assuntos
Aminoácidos , Fator Tu de Elongação de Peptídeos , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/genética , Fator Tu de Elongação de Peptídeos/metabolismo , Aminoácidos/genética , Ribossomos/metabolismo , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo
2.
PLoS Genet ; 19(1): e1010577, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36626369

RESUMO

As ribosomes translate the genetic code, they can encounter a variety of obstacles that hinder their progress. If ribosomes stall for prolonged times, cells suffer due to the loss of translating ribosomes and the accumulation of aberrant protein products. Thus to protect cells, stalled ribosomes experience a series of reactions to relieve the stall and degrade the offending mRNA, a process known as No-Go mRNA Decay (NGD). While much of the machinery for NGD is known, the precise ordering of events and factors along this pathway has not been tested. Here, we deploy C. elegans to unravel the coordinated events comprising NGD. Utilizing a novel reporter and forward and reverse genetics, we identify the machinery required for NGD. Our subsequent molecular analyses define a functional requirement for ubiquitination on at least two ribosomal proteins (eS10 and uS10), and we show that ribosomes lacking ubiquitination sites on eS10 and uS10 fail to perform NGD in vivo. We show that the nuclease NONU-1 acts after the ubiquitin ligase ZNF-598, and discover a novel requirement for the ribosome rescue factors HBS-1/PELO-1 in mRNA decay via NONU-1. Taken together, our work demonstrates mechanisms by which ribosomes signal to effectors of mRNA repression, and we delineate links between repressive factors working toward a well-defined NGD pathway.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Animais , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Caenorhabditis elegans/genética , Ribossomos/genética , Ubiquitinação , Estabilidade de RNA/genética , RNA Mensageiro/genética , Biossíntese de Proteínas
3.
Cell ; 186(2): 346-362.e17, 2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36638793

RESUMO

Ribosomes frequently stall during mRNA translation, resulting in the context-dependent activation of quality control pathways to maintain proteostasis. However, surveillance mechanisms that specifically respond to stalled ribosomes with an occluded A site have not been identified. We discovered that the elongation factor-1α (eEF1A) inhibitor, ternatin-4, triggers the ubiquitination and degradation of eEF1A on stalled ribosomes. Using a chemical genetic approach, we unveiled a signaling network comprising two E3 ligases, RNF14 and RNF25, which are required for eEF1A degradation. Quantitative proteomics revealed the RNF14 and RNF25-dependent ubiquitination of eEF1A and a discrete set of ribosomal proteins. The ribosome collision sensor GCN1 plays an essential role by engaging RNF14, which directly ubiquitinates eEF1A. The site-specific, RNF25-dependent ubiquitination of the ribosomal protein RPS27A/eS31 provides a second essential signaling input. Our findings illuminate a ubiquitin signaling network that monitors the ribosomal A site and promotes the degradation of stalled translation factors, including eEF1A and the termination factor eRF1.


Assuntos
Proteínas de Ligação a RNA , Transativadores , Proteínas de Transporte/metabolismo , Fatores de Alongamento de Peptídeos/genética , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Humanos , Células HeLa , Células HEK293 , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismo
4.
Funct Integr Genomics ; 23(1): 45, 2023 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-36683116

RESUMO

Recent studies have confirmed the direct role of vitamin B12 (VitB12) in the central nervous system (CNS) homeostasis; nevertheless, the detailed mechanisms are poorly understood. By analyzing RNA-Seq and microarray datasets obtained from databanks, this study aims to identify possible basic mechanisms, related to the brain, involved in altering the gene expression under VitB12 deficiency mimicking conditions. The database inquiry returned datasets generated from distinctly heterogeneous experimental sets and considering the quality and relevance requirements, two datasets from mouse and one from rat models were selected. The analyses of individual datasets highlighted a change in ribosomal gene expression in VitB12 deficiency mimicking conditions within each system. Specifically, a divergent regulation was observed depending on the animal model: mice showed a down regulation of the ribosomal gene expression, while rats an upregulation. Interestingly, E2f1 was significantly upregulated under VitB12 deficiency mimicking conditions in the animal models, with a greater upregulation in rats. The rat model also revealed putative E2F1 Transcription Factor Binding Sites (TFBSs) in the promoter of the differently regulated genes involved in ribosomal gene expression. This suggested the possibility that E2F1, being greater expressed in rats, could activate the ribosomal genes having E2F1 TFBSs, thus giving a plausible explication to the divergent regulation observed in animal models. Despite the great diversity of the experimental sets used to generate the datasets considered, a common alteration of the ribosomes exists, thereby indicating a possible basic and conserved response to VitB12 deficiency. Moreover, these findings could provide new insights on E2F1 and its association with CNS homeostasis and VitB12 deficiency.


Assuntos
Deficiência de Vitamina B 12 , Vitamina B 12 , Ratos , Animais , Camundongos , Vitamina B 12/genética , Vitamina B 12/metabolismo , Deficiência de Vitamina B 12/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Sistema Nervoso Central/metabolismo , Expressão Gênica
5.
Nat Commun ; 14(1): 30, 2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36596788

RESUMO

The mitochondrial translation machinery highly diverged from its bacterial counterpart. This includes deviation from the universal genetic code, with AGA and AGG codons lacking cognate tRNAs in human mitochondria. The locations of these codons at the end of COX1 and ND6 open reading frames, respectively, suggest they might function as stop codons. However, while the canonical stop codons UAA and UAG are known to be recognized by mtRF1a, the release mechanism at AGA and AGG codons remains a debated issue. Here, we show that upon the loss of another member of the mitochondrial release factor family, mtRF1, mitoribosomes accumulate specifically at AGA and AGG codons. Stalling of mitoribosomes alters COX1 transcript and protein levels, but not ND6 synthesis. In addition, using an in vitro reconstituted mitochondrial translation system, we demonstrate the specific peptide release activity of mtRF1 at the AGA and AGG codons. Together, our results reveal the role of mtRF1 in translation termination at non-canonical stop codons in mitochondria.


Assuntos
Códon de Terminação , Mitocôndrias , Fatores de Terminação de Peptídeos , Humanos , Códon de Terminação/metabolismo , Mitocôndrias/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo
6.
Signal Transduct Target Ther ; 8(1): 15, 2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36617563

RESUMO

The ribosome is a multi-unit complex that translates mRNA into protein. Ribosome biogenesis is the process that generates ribosomes and plays an essential role in cell proliferation, differentiation, apoptosis, development, and transformation. The mTORC1, Myc, and noncoding RNA signaling pathways are the primary mediators that work jointly with RNA polymerases and ribosome proteins to control ribosome biogenesis and protein synthesis. Activation of mTORC1 is required for normal fetal growth and development and tissue regeneration after birth. Myc is implicated in cancer development by enhancing RNA Pol II activity, leading to uncontrolled cancer cell growth. The deregulation of noncoding RNAs such as microRNAs, long noncoding RNAs, and circular RNAs is involved in developing blood, neurodegenerative diseases, and atherosclerosis. We review the similarities and differences between eukaryotic and bacterial ribosomes and the molecular mechanism of ribosome-targeting antibiotics and bacterial resistance. We also review the most recent findings of ribosome dysfunction in COVID-19 and other conditions and discuss the consequences of ribosome frameshifting, ribosome-stalling, and ribosome-collision. We summarize the role of ribosome biogenesis in the development of various diseases. Furthermore, we review the current clinical trials, prospective vaccines for COVID-19, and therapies targeting ribosome biogenesis in cancer, cardiovascular disease, aging, and neurodegenerative disease.


Assuntos
COVID-19 , Neoplasias , Doenças Neurodegenerativas , Humanos , Gravidez , Feminino , Vacinas contra COVID-19/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , COVID-19/metabolismo , Ribossomos/genética , Proteínas Ribossômicas/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , RNA não Traduzido , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo
7.
Mol Cell ; 83(1): 9-11, 2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36608672

RESUMO

Wang et al. (2022)1 employ real-time single-molecule fluorescence spectroscopy to monitor eukaryotic translation initiation events, revealing that, while mRNA engagement by ribosomal 43S subunits is slow, the subsequent mRNA scanning process is rapid- ∼10 times faster than translation.


Assuntos
Biossíntese de Proteínas , Ribossomos , Códon de Iniciação/genética , Ribossomos/genética , Ribossomos/metabolismo , RNA Mensageiro/metabolismo , Iniciação Traducional da Cadeia Peptídica
8.
J Chem Phys ; 158(1): 015102, 2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36610950

RESUMO

The ribosomal exit tunnel is the primary structure affecting the release of nascent proteins at the ribosome. The ribosomal exit tunnels from different species have elements of conservation and differentiation in structural and physico-chemical properties. In this study, by simulating the elongation and escape processes of nascent proteins at the ribosomal exit tunnels of four different organisms, we show that the escape process has conserved mechanisms across the domains of life. Specifically, it is found that the escape process of proteins follows the diffusion mechanism given by a simple diffusion model, and the median escape time positively correlates with the number of hydrophobic residues and the net charge of a protein for all the exit tunnels considered. These properties hold for 12 distinct proteins considered in two slightly different and improved Go-like models. It is also found that the differences in physico-chemical properties of the tunnels lead to quantitative differences in the protein escape times. In particular, the relatively strong hydrophobicity of E. coli's tunnel and the unusually high number of negatively charged amino acids on the tunnel's surface of H. marismortui lead to substantially slower escapes of proteins at these tunnels than at those of S. cerevisiae and H. sapiens.


Assuntos
Escherichia coli , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Escherichia coli/metabolismo , Ribossomos/química , Proteínas/química , Aminoácidos/química , Biossíntese de Proteínas
9.
Nat Commun ; 14(1): 300, 2023 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-36653366

RESUMO

Historically, ribosomes were viewed as unchanged homogeneous macromolecular machines with no regulatory capacity for mRNA translation. An emerging concept is that heterogeneity of ribosomal composition exists, exerting a regulatory function or specificity in translational control. This is supported by recent discoveries identifying compositionally distinct specialised ribosomes that actively regulate mRNA translation. Viruses lack their own translational machinery and impose high translational demands on the host during replication. We explore the possibility that KSHV manipulates ribosome biogenesis producing specialised ribosomes which preferentially translate viral transcripts. Quantitative proteomic analysis identified changes in the stoichiometry and composition of precursor ribosomal complexes during the switch from latent to lytic replication. We demonstrate the enhanced association of ribosomal biogenesis factors BUD23 and NOC4L, and the KSHV ORF11 protein, with small ribosomal subunit precursor complexes during lytic replication. BUD23 depletion resulted in significantly reduced viral gene expression, culminating in dramatic reduction of infectious virion production. Ribosome profiling demonstrated BUD23 is essential for reduced association of ribosomes with KSHV uORFs in late lytic genes, required for the efficient translation of the downstream coding sequence. Results provide mechanistic insights into KSHV-mediated manipulation of cellular ribosome composition inducing a population of specialised ribosomes facilitating efficient translation of viral mRNAs.


Assuntos
Herpesvirus Humano 8 , Herpesvirus Humano 8/genética , Replicação Viral/genética , Proteômica , Ribossomos/genética , Regulação Viral da Expressão Gênica
10.
Nature ; 613(7945): 712-720, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36653451

RESUMO

Ribosomes are produced in large quantities during oogenesis and are stored in the egg. However, the egg and early embryo are translationally repressed1-4. Here, using mass spectrometry and cryo-electron microscopy analyses of ribosomes isolated from zebrafish (Danio rerio) and Xenopus laevis eggs and embryos, we provide molecular evidence that ribosomes transition from a dormant state to an active state during the first hours of embryogenesis. Dormant ribosomes are associated with four conserved factors that form two modules, consisting of Habp4-eEF2 and death associated protein 1b (Dap1b) or Dap in complex with eIF5a. Both modules occupy functionally important sites and act together to stabilize ribosomes and repress translation. Dap1b (also known as Dapl1 in mammals) is a newly discovered translational inhibitor that stably inserts into the polypeptide exit tunnel. Addition of recombinant zebrafish Dap1b protein is sufficient to block translation and reconstitute the dormant egg ribosome state in a mammalian translation extract in vitro. Thus, a developmentally programmed, conserved ribosome state has a key role in ribosome storage and translational repression in the egg.


Assuntos
Ribossomos , Peixe-Zebra , Animais , Peixe-Zebra/metabolismo , Microscopia Crioeletrônica/métodos , Ribossomos/metabolismo , Peptídeos/metabolismo , Mamíferos/metabolismo , Biossíntese de Proteínas
11.
Toxins (Basel) ; 15(1)2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36668855

RESUMO

After more than 50 years of research, studies on the structure and biological activities of ribosome-inactivating proteins (RIPs) continue to provide a field of great interest within the scientific community, both for the health risks they pose and their applications in medicine and biotechnology [...].


Assuntos
Proteínas Inativadoras de Ribossomos , Ribossomos , Proteínas Inativadoras de Ribossomos/química , Ribossomos/metabolismo , Proteínas de Plantas/metabolismo
12.
Cells ; 12(2)2023 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-36672194

RESUMO

Upon oxidative stress, mammalian cells rapidly reprogram their translation. This is accompanied by the formation of stress granules (SGs), cytoplasmic ribonucleoprotein condensates containing untranslated mRNA molecules, RNA-binding proteins, 40S ribosomal subunits, and a set of translation initiation factors. Here we show that arsenite-induced stress causes a dramatic increase in the stop-codon readthrough rate and significantly elevates translation reinitiation levels on uORF-containing and bicistronic mRNAs. We also report the recruitment of translation termination factors eRF1 and eRF3, as well as ribosome recycling and translation reinitiation factors ABCE1, eIF2D, MCT-1, and DENR to SGs upon arsenite treatment. Localization of these factors to SGs may contribute to a rapid resumption of mRNA translation after stress relief and SG disassembly. It may also suggest the presence of post-termination, recycling, or reinitiation complexes in SGs. This new layer of translational control under stress conditions, relying on the altered spatial distribution of translation factors between cellular compartments, is discussed.


Assuntos
Arsenitos , Animais , Códon de Terminação , Arsenitos/farmacologia , Arsenitos/metabolismo , Ribossomos/metabolismo , Grânulos de Estresse , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Oxidativo , Mamíferos/metabolismo
13.
Nat Commun ; 14(1): 79, 2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36627279

RESUMO

The collision sensor Hel2 specifically recognizes colliding ribosomes and ubiquitinates the ribosomal protein uS10, leading to noncanonical subunit dissociation by the ribosome-associated quality control trigger (RQT) complex. Although uS10 ubiquitination is essential for rescuing stalled ribosomes, its function and recognition steps are not fully understood. Here, we show that the RQT complex components Cue3 and Rqt4 interact with the K63-linked ubiquitin chain and accelerate the recruitment of the RQT complex to the ubiquitinated colliding ribosome. The CUE domain of Cue3 and the N-terminal domain of Rqt4 bind independently to the K63-linked ubiquitin chain. Their deletion abolishes ribosomal dissociation mediated by the RQT complex. High-speed atomic force microscopy (HS-AFM) reveals that the intrinsically disordered regions of Rqt4 enable the expansion of the searchable area for interaction with the ubiquitin chain. These findings provide mechanistic insight into the decoding of the ubiquitin code for clearance of colliding ribosomes by the RQT complex.


Assuntos
Proteínas de Saccharomyces cerevisiae , Ubiquitina , Ubiquitina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ribossomos/metabolismo , Ubiquitinação , Biossíntese de Proteínas , Ubiquitina-Proteína Ligases/metabolismo
14.
PLoS Biol ; 21(1): e3001942, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36603027

RESUMO

RNA processing and degradation shape the transcriptome by generating stable molecules that are necessary for translation (rRNA and tRNA) and by facilitating the turnover of mRNA, which is necessary for the posttranscriptional control of gene expression. In bacteria and the plant chloroplast, RNA degradosomes are multienzyme complexes that process and degrade RNA. In many bacterial species, the endoribonuclease RNase E is the central component of the RNA degradosome. RNase E-based RNA degradosomes are inner membrane proteins in a large family of gram-negative bacteria (ß- and γ-Proteobacteria). Until now, the reason for membrane localization was not understood. Here, we show that a mutant strain of Escherichia coli, in which the RNA degradosome is localized to the interior of the cell, has high levels of 20S and 40S particles that are defective intermediates in ribosome assembly. These particles have aberrant protein composition and contain rRNA precursors that have been cleaved by RNase E. After RNase E cleavage, rRNA fragments are degraded to nucleotides by exoribonucleases. In vitro, rRNA in intact ribosomes is resistant to RNase E cleavage, whereas protein-free rRNA is readily degraded. We conclude that RNA degradosomes in the nucleoid of the mutant strain interfere with cotranscriptional ribosome assembly. We propose that membrane-attached RNA degradosomes in wild-type cells control the quality of ribosome assembly after intermediates are released from the nucleoid. That is, the compact structure of mature ribosomes protects rRNA against cleavage by RNase E. Turnover of a proportion of intermediates in ribosome assembly explains slow growth of the mutant strain. Competition between mRNA and rRNA degradation could be the cause of slower mRNA degradation in the mutant strain. We conclude that attachment of the RNA degradosome to the bacterial inner cytoplasmic membrane prevents wasteful degradation of rRNA precursors, thus explaining the reason for conservation of membrane-attached RNA degradosomes throughout the ß- and γ-Proteobacteria.


Assuntos
Proteínas de Escherichia coli , RNA Ribossômico , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Ribossomos/metabolismo , Complexos Multienzimáticos/metabolismo , RNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Membrana Celular/metabolismo , Bactérias/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Bacteriano/genética
15.
J Am Chem Soc ; 145(2): 851-863, 2023 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-36603206

RESUMO

Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis. To ensure compound supply for further development, we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by the development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing, and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. These studies open avenues for structure-based scaffold improvement toward development as an antibacterial agent.


Assuntos
Mycobacterium tuberculosis , Myxococcales , Antibacterianos/química , Ribossomos/metabolismo , Biossíntese de Proteínas
16.
Biochem Biophys Res Commun ; 641: 110-115, 2023 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-36527744

RESUMO

Despite the similarity in fundamental goals of translation initiation between different domains of life, it is one of the most phylogenetically diverse steps of the central dogma of molecular biology. In a classical view, the translation signals for prokaryotes and eukaryotes are distinct from each other. This idea was challenged by the finding that the Internal Ribosome Entry Site (IRES) belonging to Plautia stali intestine virus (PSIV) could bypass the domain-specific boundaries and effectively initiate translation in E. coli. This finding led us to investigate whether the ability of PSIV IRES to initiate translation in E. coli is specific to this IRES and also to study features that allow this viral IRES to mediate prokaryotic translation initiation. We observed that certain IRESs may also possess the ability to initiate E. coli translation. Our results also indicated that the structural integrity of the PSIV IRES in translation in prokaryotes does not appear to be as critical as it is in eukaryotes. We also demonstrated that two regions of the PSIV IRES with complementarity to 16S ribosomal RNA are important for the ability of this IRES to initiate translation in E. coli.


Assuntos
Sítios Internos de Entrada Ribossomal , Ribossomos , Sequência de Bases , Ribossomos/metabolismo , Sítios Internos de Entrada Ribossomal/genética , RNA Viral/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Biossíntese de Proteínas
17.
Structure ; 31(1): 4-19, 2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36584678

RESUMO

Molecular machines, such as polymerases, ribosomes, or proteasomes, fulfill complex tasks requiring the thermal energy of their environment. They achieve this by restricting random motion along a path of possible conformational changes. These changes are often directed through engagement with different cofactors, which can best be compared to a Brownian ratchet. Many molecular machines undergo three major steps throughout their functional cycles, including initialization, repetitive processing, and termination. Several of these major states have been elucidated by cryogenic electron microscopy (cryo-EM). However, the individual steps for these machines are unique and multistep processes themselves, and their coordination in time is still elusive. To measure these short-lived intermediate events by cryo-EM, the total reaction time needs to be shortened to enrich for the respective pre-equilibrium states. This approach is termed time-resolved cryo-EM (trEM). In this review, we sum up the methodological development of trEM and its application to a range of biological questions.


Assuntos
Simulação de Dinâmica Molecular , Ribossomos , Microscopia Crioeletrônica , Movimento (Física) , Tempo
18.
Nat Struct Mol Biol ; 30(1): 91-98, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36536102

RESUMO

RNA modifications are widespread in biology and abundant in ribosomal RNA. However, the importance of these modifications is not well understood. We show that methylation of a single nucleotide, in the catalytic center of the large subunit, gates ribosome assembly. Massively parallel mutational scanning of the essential nuclear GTPase Nog2 identified important interactions with rRNA, particularly with the 2'-O-methylated A-site base Gm2922. We found that methylation of G2922 is needed for assembly and efficient nuclear export of the large subunit. Critically, we identified single amino acid changes in Nog2 that completely bypass dependence on G2922 methylation and used cryoelectron microscopy to directly visualize how methylation flips Gm2922 into the active site channel of Nog2. This work demonstrates that a single RNA modification is a critical checkpoint in ribosome biogenesis, suggesting that such modifications can play an important role in regulation and assembly of macromolecular machines.


Assuntos
RNA Ribossômico , Ribossomos , RNA Ribossômico/metabolismo , Metilação , Microscopia Crioeletrônica , Ribossomos/metabolismo , Núcleo Celular/metabolismo
19.
J Biol Chem ; 299(1): 102795, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36528064

RESUMO

Shiga toxin 2a (Stx2a) is the virulence factor of enterohemorrhagic Escherichia coli. The catalytic A1 subunit of Stx2a (Stx2A1) interacts with the ribosomal P-stalk for loading onto the ribosome and depurination of the sarcin-ricin loop, which halts protein synthesis. Because of the intrinsic flexibility of the P-stalk, a structure of the Stx2a-P-stalk complex is currently unknown. We demonstrated that the native P-stalk pentamer binds to Stx2a with nanomolar affinity, and we employed cryo-EM to determine a structure of the 72 kDa Stx2a complexed with the P-stalk. The structure identifies Stx2A1 residues involved in binding and reveals that Stx2a is anchored to the P-stalk via only the last six amino acids from the C-terminal domain of a single P-protein. For the first time, the cryo-EM structure shows the loop connecting Stx2A1 and Stx2A2, which is critical for activation of the toxin. Our principal component analysis of the cryo-EM data reveals the intrinsic dynamics of the Stx2a-P-stalk interaction, including conformational changes in the P-stalk binding site occurring upon complex formation. Our computational analysis unveils the propensity for structural rearrangements within the C-terminal domain, with its C-terminal six amino acids transitioning from a random coil to an α-helix upon binding to Stx2a. In conclusion, our cryo-EM structure sheds new light into the dynamics of the Stx2a-P-stalk interaction and indicates that the binding interface between Stx2a and the P-stalk is the potential target for drug discovery.


Assuntos
Ribossomos , Toxina Shiga II , Toxina Shiga II/metabolismo , Microscopia Crioeletrônica , Ribossomos/metabolismo , Toxina Shiga/metabolismo , Aminoácidos/metabolismo
20.
Nucleic Acids Res ; 51(1): 304-314, 2023 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-36533511

RESUMO

A stop codon entering the ribosome A-site is normally decoded by release factors that induce release of the polypeptide. Certain factors influence the efficiency of the termination which is in competition with elongation in either the same (readthrough) or an alternative (frameshifting) reading frame. To gain insight into the competition between these processes, we monitored translation in parallel from all three reading frames downstream of stop codons while changing the nucleotide context of termination sites or altering cellular conditions (polyamine levels). We found that P-site codon identity can have a major impact on the termination efficiency of the OPRL1 stop signal, whereas for the OAZ1 ORF1 stop signal, the P-site codon mainly influences the reading frame of non-terminating ribosomes. Changes to polyamine levels predominantly influence the termination efficiency of the OAZ1 ORF1 stop signal. In contrast, increasing polyamine levels stimulate readthrough of the OPRL1 stop signal by enhancing near-cognate decoding rather than by decreasing termination efficiency. Thus, by monitoring the four competing processes occurring at stop codons we were able to determine which is the most significantly affected upon perturbation. This approach may be useful for the interrogation of other recoding phenomena where alternative decoding processes compete with standard decoding.


Assuntos
Códon de Terminação , Biossíntese de Proteínas , Fases de Leitura , Códon de Terminação/metabolismo , Nucleotídeos/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
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