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1.
Nat Commun ; 12(1): 5340, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504068

RESUMO

Free L-tryptophan (L-Trp) stalls ribosomes engaged in the synthesis of TnaC, a leader peptide controlling the expression of the Escherichia coli tryptophanase operon. Despite extensive characterization, the molecular mechanism underlying the recognition and response to L-Trp by the TnaC-ribosome complex remains unknown. Here, we use a combined biochemical and structural approach to characterize a TnaC variant (R23F) with greatly enhanced sensitivity for L-Trp. We show that the TnaC-ribosome complex captures a single L-Trp molecule to undergo termination arrest and that nascent TnaC prevents the catalytic GGQ loop of release factor 2 from adopting an active conformation at the peptidyl transferase center. Importantly, the L-Trp binding site is not altered by the R23F mutation, suggesting that the relative rates of L-Trp binding and peptidyl-tRNA cleavage determine the tryptophan sensitivity of each variant. Thus, our study reveals a strategy whereby a nascent peptide assists the ribosome in detecting a small metabolite.


Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Iniciação Traducional da Cadeia Peptídica , Ribossomos/genética , Triptofano/química , Substituição de Aminoácidos , Sítios de Ligação , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Mutação , Óperon , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Triptofano/metabolismo
2.
Nat Commun ; 12(1): 4909, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389707

RESUMO

In bacteria, trans-translation is the main rescue system, freeing ribosomes stalled on defective messenger RNAs. This mechanism is driven by small protein B (SmpB) and transfer-messenger RNA (tmRNA), a hybrid RNA known to have both a tRNA-like and an mRNA-like domain. Here we present four cryo-EM structures of the ribosome during trans-translation at resolutions from 3.0 to 3.4 Å. These include the high-resolution structure of the whole pre-accommodated state, as well as structures of the accommodated state, the translocated state, and a translocation intermediate. Together, they shed light on the movements of the tmRNA-SmpB complex in the ribosome, from its delivery by the elongation factor EF-Tu to its passage through the ribosomal A and P sites after the opening of the B1 bridges. Additionally, we describe the interactions between the tmRNA-SmpB complex and the ribosome. These explain why the process does not interfere with canonical translation.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Biossíntese de Proteínas/genética , RNA Bacteriano/genética , Proteínas de Ligação a RNA/genética , Ribossomos/genética , Sítios de Ligação/genética , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura
3.
Nat Commun ; 12(1): 4644, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330903

RESUMO

Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. How and where in the elongation cycle +1-frameshifting occurs remains poorly understood. We describe seven ~3.5-Å-resolution cryo-EM structures of 70S ribosome complexes, allowing visualization of elongation and translocation by the GTPase elongation factor G (EF-G). Four structures with a + 1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-G•GDPCP, the tRNA shifts to the +1-frame near the P site, rendering the freed mRNA base to bulge between the P and E sites and to stack on the 16S rRNA nucleotide G926. The ribosome remains frameshifted in the nearly post-translocation state. Our findings demonstrate that the ribosome and EF-G cooperate to induce +1 frameshifting during tRNA-mRNA translocation.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico/genética , Elongação Traducional da Cadeia Peptídica/genética , Fator G para Elongação de Peptídeos/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Ribossomos/genética , Biocatálise , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fator G para Elongação de Peptídeos/química , Fator G para Elongação de Peptídeos/metabolismo , Conformação Proteica , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismo
4.
Int J Mol Sci ; 22(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200244

RESUMO

Ribosome biogenesis is a highly coordinated and complex process that requires numerous assembly factors that ensure prompt and flawless maturation of ribosomal subunits. Despite the increasing amount of data collected, the exact role of most assembly factors and mechanistic details of their operation remain unclear, mainly due to the shortage of high-resolution structural information. Here, using cryo-electron microscopy, we characterized 30S ribosomal particles isolated from an Escherichia coli strain with a deleted gene for the RbfA factor. The cryo-EM maps for pre-30S subunits were divided into six classes corresponding to consecutive assembly intermediates: from the particles with a completely unresolved head domain and unfolded central pseudoknot to almost mature 30S subunits with well-resolved body, platform, and head domains and partially distorted helix 44. The structures of two predominant 30S intermediates belonging to most populated classes obtained at 2.7 Å resolutions indicate that RbfA acts at two distinctive 30S assembly stages: early formation of the central pseudoknot including folding of the head, and positioning of helix 44 in the decoding center at a later stage. Additionally, it was shown that the formation of the central pseudoknot may promote stabilization of the head domain, likely through the RbfA-dependent maturation of the neck helix 28. An update to the model of factor-dependent 30S maturation is proposed, suggesting that RfbA is involved in most of the subunit assembly process.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Bactérias/fisiologia , Ribossomos/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica/métodos , Proteínas de Escherichia coli/genética , Modelos Moleculares , Ligação Proteica , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Menores de Bactérias/ultraestrutura , Ribossomos/ultraestrutura
5.
Nat Commun ; 12(1): 3850, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158503

RESUMO

Three stop codons (UAA, UAG and UGA) terminate protein synthesis and are almost exclusively recognized by release factors. Here, we design de novo transfer RNAs (tRNAs) that efficiently decode UGA stop codons in Escherichia coli. The tRNA designs harness various functionally conserved aspects of sense-codon decoding tRNAs. Optimization within the TΨC-stem to stabilize binding to the elongation factor, displays the most potent effect in enhancing suppression activity. We determine the structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon in the A site at 2.9 Å resolution. In the context of the suppressor tRNA, the conformation of the UGA codon resembles that of a sense-codon rather than when canonical translation termination release factors are bound, suggesting conformational flexibility of the stop codons dependent on the nature of the A-site ligand. The systematic analysis, combined with structural insights, provides a rationale for targeted repurposing of tRNAs to correct devastating nonsense mutations that introduce a premature stop codon.


Assuntos
Códon sem Sentido/genética , Códon de Terminação/genética , Escherichia coli/genética , Biossíntese de Proteínas/genética , RNA de Transferência/genética , Ribossomos/genética , Sequência de Bases , Sítios de Ligação/genética , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Supressão Genética
6.
Science ; 372(6548): 1306-1313, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34029205

RESUMO

Programmed ribosomal frameshifting is a key event during translation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome that allows synthesis of the viral RNA-dependent RNA polymerase and downstream proteins. Here, we present the cryo-electron microscopy structure of a translating mammalian ribosome primed for frameshifting on the viral RNA. The viral RNA adopts a pseudoknot structure that lodges at the entry to the ribosomal messenger RNA (mRNA) channel to generate tension in the mRNA and promote frameshifting, whereas the nascent viral polyprotein forms distinct interactions with the ribosomal tunnel. Biochemical experiments validate the structural observations and reveal mechanistic and regulatory features that influence frameshifting efficiency. Finally, we compare compounds previously shown to reduce frameshifting with respect to their ability to inhibit SARS-CoV-2 replication, establishing coronavirus frameshifting as a target for antiviral intervention.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , RNA Viral/genética , Ribossomos/ultraestrutura , SARS-CoV-2/genética , Proteínas Virais/biossíntese , Animais , Antivirais/farmacologia , Códon de Terminação , RNA-Polimerase RNA-Dependente de Coronavírus/biossíntese , RNA-Polimerase RNA-Dependente de Coronavírus/química , RNA-Polimerase RNA-Dependente de Coronavírus/genética , Microscopia Crioeletrônica , Fluoroquinolonas/farmacologia , Mudança da Fase de Leitura do Gene Ribossômico/efeitos dos fármacos , Genoma Viral , Humanos , Processamento de Imagem Assistida por Computador , Modelos Moleculares , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Dobramento de Proteína , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico 18S/química , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , RNA Viral/química , RNA Viral/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Proteínas Virais/química , Proteínas Virais/genética , Replicação Viral/efeitos dos fármacos
7.
Methods Mol Biol ; 2305: 291-299, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33950395

RESUMO

Cryo electron microscopy (cryo-EM) has become a method of choice in structural biology to analyze isolated complexes and cellular structures. This implies adequate imaging of the specimen and advanced image-processing methods to obtain high-resolution 3D reconstructions. The use of a Volta phase plate in cryo-EM drastically increases the image contrast while being able to record images at high acceleration voltage and close to focus, i.e., at conditions where high-resolution information is best preserved. During image processing, higher contrast images can be aligned and classified better than lower quality ones resulting in increased data quality and the need for less data. Here, we give step-by-step guidelines on how to set up high-quality VPP cryo-EM single particle data collections, as exemplified by human ribosome data acquired during a one-day data collection session. Further, we describe specific technical details in image processing that differ from conventional single particle cryo-EM data analysis.


Assuntos
Microscopia Crioeletrônica/métodos , Coleta de Dados/métodos , Ribossomos/ultraestrutura , Microscopia Crioeletrônica/instrumentação , Confiabilidade dos Dados , Humanos , Imageamento Tridimensional/métodos , Biologia Molecular/métodos , Ribossomos/química , Imagem Individual de Molécula
8.
J Mol Biol ; 433(10): 166942, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33744313

RESUMO

Macrolide antibiotics, such as erythromycin, bind to the nascent peptide exit tunnel (NPET) of the bacterial ribosome and modulate protein synthesis depending on the nascent peptide sequence. Whereas in vitro biochemical and structural methods have been instrumental in dissecting and explaining the molecular details of macrolide-induced peptidyl-tRNA drop-off and ribosome stalling, the dynamic effects of the drugs on ongoing protein synthesis inside live bacterial cells are far less explored. In the present study, we used single-particle tracking of dye-labeled tRNAs to study the kinetics of mRNA translation in the presence of erythromycin, directly inside live Escherichia coli cells. In erythromycin-treated cells, we find that the dwells of elongator tRNAPhe on ribosomes extend significantly, but they occur much more seldom. In contrast, the drug barely affects the ribosome binding events of the initiator tRNAfMet. By overexpressing specific short peptides, we further find context-specific ribosome binding dynamics of tRNAPhe, underscoring the complexity of erythromycin's effect on protein synthesis in bacterial cells.


Assuntos
Antibacterianos/farmacologia , Eritromicina/farmacologia , Escherichia coli/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Ribossomos/efeitos dos fármacos , Sequência de Aminoácidos , Antibacterianos/metabolismo , Carbocianinas/química , Códon , Eritromicina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Corantes Fluorescentes/química , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Inibidores da Síntese de Proteínas/metabolismo , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA de Transferência de Metionina/química , RNA de Transferência de Metionina/genética , RNA de Transferência de Metionina/metabolismo , RNA de Transferência de Fenilalanina/química , RNA de Transferência de Fenilalanina/genética , RNA de Transferência de Fenilalanina/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Imagem Individual de Molécula
9.
Nat Chem Biol ; 17(4): 412-420, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33462493

RESUMO

Many antibiotics inhibit bacterial growth by binding to the ribosome and interfering with protein biosynthesis. Macrolides represent one of the most successful classes of ribosome-targeting antibiotics. The main clinically relevant mechanism of resistance to macrolides is dimethylation of the 23S rRNA nucleotide A2058, located in the drug-binding site, a reaction catalyzed by Erm-type rRNA methyltransferases. Here, we present the crystal structure of the Erm-dimethylated 70S ribosome at 2.4 Å resolution, together with the structures of unmethylated 70S ribosome functional complexes alone or in combination with macrolides. Altogether, our structural data do not support previous models and, instead, suggest a principally new explanation of how A2058 dimethylation confers resistance to macrolides. Moreover, high-resolution structures of two macrolide antibiotics bound to the unmodified ribosome reveal a previously unknown role of the desosamine moiety in drug binding, laying a foundation for the rational knowledge-based design of macrolides that can overcome Erm-mediated resistance.


Assuntos
Macrolídeos/metabolismo , RNA Ribossômico/ultraestrutura , Ribossomos/ultraestrutura , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Macrolídeos/farmacologia , Metilação , RNA Ribossômico/genética , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , RNA Ribossômico 23S/ultraestrutura , Ribossomos/genética , Ribossomos/metabolismo
10.
FEBS J ; 288(2): 663-677, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32337775

RESUMO

Canonically, tRNA synthetases charge tRNA. However, the lysyl-tRNA synthetase paralog EpmA catalyzes the attachment of (R)-ß-lysine to the ε-amino group of lysine 34 of the translation elongation factor P (EF-P) in Escherichia coli. This modification is essential for EF-P-mediated translational rescue of ribosomes stalled at consecutive prolines. In this study, we determined the kinetics of EpmA and its variant EpmA_A298G to catalyze the post-translational modification of K34 in EF-P with eight noncanonical substrates. In addition, acetylated EF-P was generated using an amber suppression system. The impact of these synthetically modified EF-P variants on in vitro translation of a polyproline-containing NanoLuc luciferase reporter was analyzed. Our results show that natural (R)-ß-lysylation was more effective in rescuing stalled ribosomes than any other synthetic modification tested. Thus, our work not only provides new biochemical insights into the function of EF-P, but also opens a new route to post-translationally modify proteins using EpmA.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Lisina-tRNA Ligase/genética , Fatores de Alongamento de Peptídeos/genética , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Acetilação , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Genes Reporter , Cinética , Luciferases/genética , Luciferases/metabolismo , Lisina/genética , Lisina/metabolismo , Lisina-tRNA Ligase/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Mutação Puntual , Prolina/genética , Prolina/metabolismo , RNA de Transferência de Lisina/genética , RNA de Transferência de Lisina/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Especificidade por Substrato
11.
Subcell Biochem ; 96: 433-450, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33252739

RESUMO

Expansion segments (ES) are insertions of a few to hundreds of nucleotides at discrete locations on eukaryotic ribosomal RNA (rRNA) chains. Some cluster around 'hot spots' involved in translation regulation and some may participate in biogenesis. Whether ES play the same roles in different organisms is currently unclear, especially since their size may vary dramatically from one species to another and very little is known about their functions. Most likely, ES variation is linked to adaptation to a particular environment. In this chapter, we compare the interaction networks of ES from four kinetoplastid parasites, which have evolved in diverse insect vectors and mammalian hosts: Trypanosoma cruzi, Trypanosoma brucei, Leishmania donovani and Leishmania major. Here, we comparatively analyze ribosome structures from these representative kinetoplastids and ascertain meaningful structural differences from mammalian ribosomes. We base our analysis on sequence alignments and three-dimensional structures of 80S ribosomes solved by cryo-electron microscopy (cryo-EM). Striking differences in size are observed between ribosomes of different parasites, indicating that not all ES are expanded equally. Larger ES are not always matched by large surrounding ES or proteins extensions in their vicinity, a particularity that may lead to clues about their biological function. ES display different species-specific patterns of conservation, which underscore the density of their interaction network at the surface of the ribosome. Making sense of the conservation patterns of ES is part of a global effort to lay the basis for functional studies aimed at discovering unique kinetoplastid-specific sites suitable for therapeutic applications against these human and often animal pathogens.


Assuntos
Kinetoplastida/genética , RNA Ribossômico/genética , Ribossomos/metabolismo , Animais , Microscopia Crioeletrônica , Células Eucarióticas/metabolismo , Humanos , Kinetoplastida/patogenicidade , Ribossomos/química , Ribossomos/ultraestrutura
12.
Nucleic Acids Res ; 49(1): 547-567, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33330920

RESUMO

Genomic studies have indicated that certain bacterial lineages such as the Bacteroidetes lack Shine-Dalgarno (SD) sequences, and yet with few exceptions ribosomes of these organisms carry the canonical anti-SD (ASD) sequence. Here, we show that ribosomes purified from Flavobacterium johnsoniae, a representative of the Bacteroidetes, fail to recognize the SD sequence of mRNA in vitro. A cryo-electron microscopy structure of the complete 70S ribosome from F. johnsoniae at 2.8 Å resolution reveals that the ASD is sequestered by ribosomal proteins bS21, bS18 and bS6, explaining the basis of ASD inhibition. The structure also uncovers a novel ribosomal protein-bL38. Remarkably, in F. johnsoniae and many other Flavobacteriia, the gene encoding bS21 contains a strong SD, unlike virtually all other genes. A subset of Flavobacteriia have an alternative ASD, and in these organisms the fully complementary sequence lies upstream of the bS21 gene, indicative of natural covariation. In other Bacteroidetes classes, strong SDs are frequently found upstream of the genes for bS21 and/or bS18. We propose that these SDs are used as regulatory elements, enabling bS21 and bS18 to translationally control their own production.


Assuntos
Bacteroidetes/genética , Iniciação Traducional da Cadeia Peptídica , Sequências Reguladoras de Ácido Ribonucleico , Ribossomos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Códon de Iniciação , Microscopia Crioeletrônica , Cristalografia por Raios X , Escherichia coli/genética , Flavobacterium/genética , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Puromicina/farmacologia , RNA Bacteriano/genética , RNA Mensageiro/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , RNA Ribossômico 5S/genética , Ribossomos/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência , Especificidade da Espécie
13.
Nucleic Acids Res ; 49(1): 206-220, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33330942

RESUMO

Proteostasis needs to be tightly controlled to meet the cellular demand for correctly de novo folded proteins and to avoid protein aggregation. While a coupling between translation rate and co-translational folding, likely involving an interplay between the ribosome and its associated chaperones, clearly appears to exist, the underlying mechanisms and the contribution of ribosomal proteins remain to be explored. The ribosomal protein uL3 contains a long internal loop whose tip region is in close proximity to the ribosomal peptidyl transferase center. Intriguingly, the rpl3[W255C] allele, in which the residue making the closest contact to this catalytic site is mutated, affects diverse aspects of ribosome biogenesis and function. Here, we have uncovered, by performing a synthetic lethal screen with this allele, an unexpected link between translation and the folding of nascent proteins by the ribosome-associated Ssb-RAC chaperone system. Our results reveal that uL3 and Ssb-RAC cooperate to prevent 80S ribosomes from piling up within the 5' region of mRNAs early on during translation elongation. Together, our study provides compelling in vivo evidence for a functional connection between peptide bond formation at the peptidyl transferase center and chaperone-assisted de novo folding of nascent polypeptides at the solvent-side of the peptide exit tunnel.


Assuntos
Chaperonas Moleculares/fisiologia , Complexos Multiproteicos/fisiologia , Elongação Traducional da Cadeia Peptídica/fisiologia , Dobramento de Proteína , Proteostase/fisiologia , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Alelos , Mutação com Perda de Função , Chaperonas Moleculares/genética , Mutação de Sentido Incorreto , Peptidil Transferases/fisiologia , Mutação Puntual , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/fisiologia , Ribossomos/ultraestrutura , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
14.
Mol Cell ; 81(2): 293-303.e4, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33326748

RESUMO

Ribosome assembly is catalyzed by numerous trans-acting factors and coupled with irreversible pre-rRNA processing, driving the pathway toward mature ribosomal subunits. One decisive step early in this progression is removal of the 5' external transcribed spacer (5'-ETS), an RNA extension at the 18S rRNA that is integrated into the huge 90S pre-ribosome structure. Upon endo-nucleolytic cleavage at an internal site, A1, the 5'-ETS is separated from the 18S rRNA and degraded. Here we present biochemical and cryo-electron microscopy analyses that depict the RNA exosome, a major 3'-5' exoribonuclease complex, in a super-complex with the 90S pre-ribosome. The exosome is docked to the 90S through its co-factor Mtr4 helicase, a processive RNA duplex-dismantling helicase, which strategically positions the exosome at the base of 5'-ETS helices H9-H9', which are dislodged in our 90S-exosome structures. These findings suggest a direct role of the exosome in structural remodeling of the 90S pre-ribosome to drive eukaryotic ribosome synthesis.


Assuntos
RNA Helicases DEAD-box/química , Endorribonucleases/química , Exonucleases/química , Complexo Multienzimático de Ribonucleases do Exossomo/ultraestrutura , RNA Ribossômico 18S/química , Ribossomos/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Sítios de Ligação , Microscopia Crioeletrônica , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Exonucleases/genética , Exonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Modelos Moleculares , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estabilidade de RNA , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
Nat Commun ; 11(1): 5552, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33144582

RESUMO

Ribosomes stalled during translation must be rescued to replenish the pool of translation-competent ribosomal subunits. Bacterial alternative rescue factor B (ArfB) releases nascent peptides from ribosomes stalled on mRNAs truncated at the A site, allowing ribosome recycling. Prior structural work revealed that ArfB recognizes such ribosomes by inserting its C-terminal α-helix into the vacant mRNA tunnel. In this work, we report that ArfB can efficiently recognize a wider range of mRNA substrates, including longer mRNAs that extend beyond the A-site codon. Single-particle cryo-EM unveils that ArfB employs two modes of function depending on the mRNA length. ArfB acts as a monomer to accommodate a shorter mRNA in the ribosomal A site. By contrast, longer mRNAs are displaced from the mRNA tunnel by more than 20 Å and are stabilized in the intersubunit space by dimeric ArfB. Uncovering distinct modes of ArfB function resolves conflicting biochemical and structural studies, and may lead to re-examination of other ribosome rescue pathways, whose functions depend on mRNA lengths.


Assuntos
Proteínas de Escherichia coli/metabolismo , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Biocatálise , Dimerização , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestrutura , Modelos Biológicos , Conformação Proteica , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/ultraestrutura , Subunidades Ribossômicas/metabolismo , Ribossomos/ultraestrutura
16.
Mol Cell ; 80(6): 980-995.e13, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33202249

RESUMO

Ribosomes have been suggested to directly control gene regulation, but regulatory roles for ribosomal RNA (rRNA) remain largely unexplored. Expansion segments (ESs) consist of multitudes of tentacle-like rRNA structures extending from the core ribosome in eukaryotes. ESs are remarkably variable in sequence and size across eukaryotic evolution with largely unknown functions. In characterizing ribosome binding to a regulatory element within a Homeobox (Hox) 5' UTR, we identify a modular stem-loop within this element that binds to a single ES, ES9S. Engineering chimeric, "humanized" yeast ribosomes for ES9S reveals that an evolutionary change in the sequence of ES9S endows species-specific binding of Hoxa9 mRNA to the ribosome. Genome editing to site-specifically disrupt the Hoxa9-ES9S interaction demonstrates the functional importance for such selective mRNA-rRNA binding in translation control. Together, these studies unravel unexpected gene regulation directly mediated by rRNA and how ribosome evolution drives translation of critical developmental regulators.


Assuntos
Proteínas de Homeodomínio/genética , Biossíntese de Proteínas/genética , RNA Ribossômico/ultraestrutura , Ribossomos/genética , Regiões 5' não Traduzidas/genética , Regulação da Expressão Gênica/genética , Genes Homeobox/genética , Proteínas de Homeodomínio/ultraestrutura , Conformação de Ácido Nucleico , RNA Mensageiro/genética , RNA Ribossômico/genética , Ribossomos/ultraestrutura , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Especificidade da Espécie
17.
Int J Mol Sci ; 21(19)2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33019721

RESUMO

Recent studies uncover that subcellular location of long non-coding RNAs (lncRNAs) can provide significant information on its function. Due to the lack of experimental data, the number of lncRNAs is very limited, experimentally verified subcellular localization, and the numbers of lncRNAs located in different organelle are wildly imbalanced. The prediction of subcellular location of lncRNAs is actually a multi-classification small sample imbalance problem. The imbalance of data results in the poor recognition effect of machine learning models on small data subsets, which is a puzzling and challenging problem in the existing research. In this study, we integrate multi-source features to construct a sequence-based computational tool, lncLocation, to predict the subcellular location of lncRNAs. Autoencoder is used to enhance part of the features, and the binomial distribution-based filtering method and recursive feature elimination (RFE) are used to filter some of the features. It improves the representation ability of data and reduces the problem of unbalanced multi-classification data. By comprehensive experiments on different feature combinations and machine learning models, we select the optimal features and classifier model scheme to construct a subcellular location prediction tool, lncLocation. LncLocation can obtain an 87.78% accuracy using 5-fold cross validation on the benchmark data, which is higher than the state-of-the-art tools, and the classification performance, especially for small class sets, is improved significantly.


Assuntos
Células Eucarióticas/metabolismo , Genoma Humano , RNA Longo não Codificante/genética , Software , Máquina de Vetores de Suporte , Animais , Sequência de Bases , Benchmarking , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Biologia Computacional/métodos , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Bases de Dados Genéticas , Conjuntos de Dados como Assunto , Células Eucarióticas/ultraestrutura , Exossomos/metabolismo , Exossomos/ultraestrutura , Regulação da Expressão Gênica , Humanos , RNA Longo não Codificante/classificação , RNA Longo não Codificante/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Terminologia como Assunto
18.
Nat Commun ; 11(1): 4941, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009412

RESUMO

Methods to directly inhibit gene expression using small molecules hold promise for the development of new therapeutics targeting proteins that have evaded previous attempts at drug discovery. Among these, small molecules including the drug-like compound PF-06446846 (PF846) selectively inhibit the synthesis of specific proteins, by stalling translation elongation. These molecules also inhibit translation termination by an unknown mechanism. Using cryo-electron microscopy (cryo-EM) and biochemical approaches, we show that PF846 inhibits translation termination by arresting the nascent chain (NC) in the ribosome exit tunnel. The arrested NC adopts a compact α-helical conformation that induces 28 S rRNA nucleotide rearrangements that suppress the peptidyl transferase center (PTC) catalytic activity stimulated by eukaryotic release factor 1 (eRF1). These data support a mechanism of action for a small molecule targeting translation that suppresses peptidyl-tRNA hydrolysis promoted by eRF1, revealing principles of eukaryotic translation termination and laying the foundation for new therapeutic strategies.


Assuntos
Terminação Traducional da Cadeia Peptídica , Preparações Farmacêuticas/metabolismo , Linhagem Celular , Humanos , Modelos Moleculares , Mutação/genética , Conformação Proteica , RNA Ribossômico/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura
19.
PLoS Biol ; 18(10): e3000958, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33125369

RESUMO

Assembling and powering ribosomes are energy-intensive processes requiring fine-tuned cellular control mechanisms. In organisms operating under strict nutrient limitations, such as pathogenic microsporidia, conservation of energy via ribosomal hibernation and recycling is critical. The mechanisms by which hibernation is achieved in microsporidia, however, remain poorly understood. Here, we present the cryo-electron microscopy structure of the ribosome from Paranosema locustae spores, bound by the conserved eukaryotic hibernation and recycling factor Lso2. The microsporidian Lso2 homolog adopts a V-shaped conformation to bridge the mRNA decoding site and the large subunit tRNA binding sites, providing a reversible ribosome inactivation mechanism. Although microsporidian ribosomes are highly compacted, the P. locustae ribosome retains several rRNA segments absent in other microsporidia, and represents an intermediate state of rRNA reduction. In one case, the near complete reduction of an expansion segment has resulted in a single bound nucleotide, which may act as an architectural co-factor to stabilize a protein-protein interface. The presented structure highlights the reductive evolution in these emerging pathogens and sheds light on a conserved mechanism for eukaryotic ribosome hibernation.


Assuntos
Microsporídios/metabolismo , Microsporídios/ultraestrutura , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Microscopia Crioeletrônica , Evolução Molecular , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Nucleotídeos/metabolismo , Ligação Proteica , Proteínas Ribossômicas/metabolismo
20.
Cell ; 182(6): 1606-1622.e23, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32888429

RESUMO

The enteric nervous system (ENS) coordinates diverse functions in the intestine but has eluded comprehensive molecular characterization because of the rarity and diversity of cells. Here we develop two methods to profile the ENS of adult mice and humans at single-cell resolution: RAISIN RNA-seq for profiling intact nuclei with ribosome-bound mRNA and MIRACL-seq for label-free enrichment of rare cell types by droplet-based profiling. The 1,187,535 nuclei in our mouse atlas include 5,068 neurons from the ileum and colon, revealing extraordinary neuron diversity. We highlight circadian expression changes in enteric neurons, show that disease-related genes are dysregulated with aging, and identify differences between the ileum and proximal/distal colon. In humans, we profile 436,202 nuclei, recovering 1,445 neurons, and identify conserved and species-specific transcriptional programs and putative neuro-epithelial, neuro-stromal, and neuro-immune interactions. The human ENS expresses risk genes for neuropathic, inflammatory, and extra-intestinal diseases, suggesting neuronal contributions to disease.


Assuntos
Sistema Nervoso Entérico/citologia , Sistema Nervoso Entérico/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Neurônios/metabolismo , Corpos de Nissl/metabolismo , RNA Mensageiro/metabolismo , Análise de Célula Única/métodos , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Relógios Circadianos/genética , Colo/citologia , Colo/metabolismo , Retículo Endoplasmático Rugoso/genética , Retículo Endoplasmático Rugoso/metabolismo , Retículo Endoplasmático Rugoso/ultraestrutura , Células Epiteliais/metabolismo , Feminino , Predisposição Genética para Doença/genética , Humanos , Íleo/citologia , Íleo/metabolismo , Inflamação/genética , Inflamação/metabolismo , Enteropatias/genética , Enteropatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Corpos de Nissl/genética , Corpos de Nissl/ultraestrutura , RNA Mensageiro/genética , RNA-Seq , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Células Estromais/metabolismo
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