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1.
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
2.
Nat Commun ; 11(1): 3279, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32606306

RESUMO

Recombinant adeno-associated viruses (rAAVs) are currently considered the safest and most reliable gene delivery vehicles for human gene therapy. Three serotype capsids, AAV1, AAV2, and AAV9, have been approved for commercial use in patients, but they may not be suitable for all therapeutic contexts. Here, we describe a novel capsid identified in a human clinical sample by high-throughput, long-read sequencing. The capsid, which we have named AAVv66, shares high sequence similarity with AAV2. We demonstrate that compared to AAV2, AAVv66 exhibits enhanced production yields, virion stability, and CNS transduction. Unique structural properties of AAVv66 visualized by cryo-EM at 2.5-Å resolution, suggest that critical residues at the three-fold protrusion and at the interface of the five-fold axis of symmetry likely contribute to the beneficial characteristics of AAVv66. Our findings underscore the potential of AAVv66 as a gene therapy vector.


Assuntos
Proteínas do Capsídeo/genética , Capsídeo/metabolismo , Dependovirus/genética , Vetores Genéticos/genética , Animais , Capsídeo/ultraestrutura , Proteínas do Capsídeo/classificação , Sistema Nervoso Central/virologia , Microscopia Crioeletrônica , DNA Viral/análise , DNA Viral/genética , Dependovirus/classificação , Dependovirus/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Filogenia , Sorogrupo , Transdução Genética , Montagem de Vírus/genética
3.
Nature ; 584(7822): 640-645, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32612237

RESUMO

Ribosomes accurately decode mRNA by proofreading each aminoacyl-tRNA that is delivered by the elongation factor EF-Tu1. To understand the molecular mechanism of this proofreading step it is necessary to visualize GTP-catalysed elongation, which has remained a challenge2-4. Here we use time-resolved cryogenic electron microscopy to reveal 33 ribosomal states after the delivery of aminoacyl-tRNA by EF-Tu•GTP. Instead of locking cognate tRNA upon initial recognition, the ribosomal decoding centre dynamically monitors codon-anticodon interactions before and after GTP hydrolysis. GTP hydrolysis enables the GTPase domain of EF-Tu to extend away, releasing EF-Tu from tRNA. The 30S subunit then locks cognate tRNA in the decoding centre and rotates, enabling the tRNA to bypass 50S protrusions during accommodation into the peptidyl transferase centre. By contrast, the decoding centre fails to lock near-cognate tRNA, enabling the dissociation of near-cognate tRNA both during initial selection (before GTP hydrolysis) and proofreading (after GTP hydrolysis). These findings reveal structural similarity between ribosomes in initial selection states5,6 and in proofreading states, which together govern the efficient rejection of incorrect tRNA.


Assuntos
Microscopia Crioeletrônica , Guanosina Trifosfato/metabolismo , Fator Tu de Elongação de Peptídeos/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Escherichia coli , GTP Fosfo-Hidrolases/metabolismo , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Hidrólise , Modelos Moleculares , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/ultraestrutura , Ribossomos/química , Rotação
4.
Nat Commun ; 11(1): 2900, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32518240

RESUMO

5S rRNA is an indispensable component of cytoplasmic ribosomes in all species. The functions of 5S rRNA and the reasons for its evolutionary preservation as an independent molecule remain unclear. Here we used ribosome engineering to investigate whether 5S rRNA autonomy is critical for ribosome function and cell survival. By linking circularly permutated 5S rRNA with 23S rRNA we generated a bacterial strain devoid of free 5S rRNA. Viability of the engineered cells demonstrates that autonomous 5S rRNA is dispensable for cell growth under standard conditions and is unlikely to have essential functions outside the ribosome. The fully assembled ribosomes carrying 23S-5S rRNA are highly active in translation. However, the engineered cells accumulate aberrant 50S subunits unable to form stable 70S ribosomes. Cryo-EM analysis revealed a malformed peptidyl transferase center in the misassembled 50S subunits. Our results argue that the autonomy of 5S rRNA is preserved due to its role in ribosome biogenesis.


Assuntos
RNA Ribossômico 5S/metabolismo , Ribossomos/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação da Expressão Gênica , Engenharia Genética , Mutação , Conformação de Ácido Nucleico , Peptidil Transferases/metabolismo , RNA Bacteriano , RNA Ribossômico 23S/metabolismo , Recombinases Rec A/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/metabolismo
5.
Elife ; 82019 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-31513010

RESUMO

Protein synthesis ends when a ribosome reaches an mRNA stop codon. Release factors (RFs) decode the stop codon, hydrolyze peptidyl-tRNA to release the nascent protein, and then dissociate to allow ribosome recycling. To visualize termination by RF2, we resolved a cryo-EM ensemble of E. coli 70S•RF2 structures at up to 3.3 Å in a single sample. Five structures suggest a highly dynamic termination pathway. Upon peptidyl-tRNA hydrolysis, the CCA end of deacyl-tRNA departs from the peptidyl transferase center. The catalytic GGQ loop of RF2 is rearranged into a long ß-hairpin that plugs the peptide tunnel, biasing a nascent protein toward the ribosome exit. Ribosomal intersubunit rotation destabilizes the catalytic RF2 domain on the 50S subunit and disassembles the central intersubunit bridge B2a, resulting in RF2 departure. Our structures visualize how local rearrangements and spontaneous inter-subunit rotation poise the newly-made protein and RF2 to dissociate in preparation for ribosome recycling.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/metabolismo , Ribossomos/metabolismo , Microscopia Crioeletrônica , Proteínas de Escherichia coli/química , Fatores de Terminação de Peptídeos/química , Ribossomos/química
6.
Methods ; 137: 55-66, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29247757

RESUMO

Bacterial ribosomal protein S1 is the largest and highly flexible protein of the 30S subunit, and one of a few core ribosomal proteins for which a complete structure is lacking. S1 is thought to participate in transcription and translation. Best understood is the role of S1 in facilitating translation of mRNAs with structured 5' UTRs. Here, we present cryo-EM analyses of the 70S ribosome that reveal multiple conformations of S1. Based on comparison of several 3D maximum likelihood classification approaches in Frealign, we propose a streamlined strategy for visualizing a highly dynamic component of a large macromolecular assembly that itself exhibits high compositional and conformational heterogeneity. The resulting maps show how S1 docks at the ribosomal protein S2 near the mRNA exit channel. The globular OB-fold domains sample a wide area around the mRNA exit channel and interact with mobile tails of proteins S6 and S18. S1 also interacts with the mRNA entrance channel, where an OB-fold domain can be localized near S3 and S5. Our analyses suggest that S1 cooperates with other ribosomal proteins to form a dynamic mesh near the mRNA exit and entrance channels to modulate the binding, folding and movement of mRNA.


Assuntos
Microscopia Crioeletrônica/métodos , RNA Ribossômico/ultraestrutura , Proteínas Ribossômicas/ultraestrutura , Subunidades Ribossômicas Maiores/ultraestrutura , Citosol/ultraestrutura , Escherichia coli/genética , Escherichia coli/ultraestrutura , Conformação Proteica , Proteínas Ribossômicas/química , Subunidades Ribossômicas Maiores/química
7.
Elife ; 62017 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-29027901

RESUMO

In bacteria, mRNA transcription and translation are coupled to coordinate optimal gene expression and maintain genome stability. Coupling is thought to involve direct interactions between RNA polymerase (RNAP) and the translational machinery. We present cryo-EM structures of E. coli RNAP core bound to the small ribosomal 30S subunit. The complex is stable under cell-like ionic conditions, consistent with functional interaction between RNAP and the 30S subunit. The RNA exit tunnel of RNAP aligns with the Shine-Dalgarno-binding site of the 30S subunit. Ribosomal protein S1 forms a wall of the tunnel between RNAP and the 30S subunit, consistent with its role in directing mRNAs onto the ribosome. The nucleic-acid-binding cleft of RNAP samples distinct conformations, suggesting different functional states during transcription-translation coupling. The architecture of the 30S•RNAP complex provides a structural basis for co-localization of the transcriptional and translational machineries, and inform future mechanistic studies of coupled transcription and translation.


Assuntos
RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Microscopia Crioeletrônica , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo
8.
Nature ; 546(7656): 113-117, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28538735

RESUMO

Gene translation depends on accurate decoding of mRNA, the structural mechanism of which remains poorly understood. Ribosomes decode mRNA codons by selecting cognate aminoacyl-tRNAs delivered by elongation factor Tu (EF-Tu). Here we present high-resolution structural ensembles of ribosomes with cognate or near-cognate aminoacyl-tRNAs delivered by EF-Tu. Both cognate and near-cognate tRNA anticodons explore the aminoacyl-tRNA-binding site (A site) of an open 30S subunit, while inactive EF-Tu is separated from the 50S subunit. A transient conformation of decoding-centre nucleotide G530 stabilizes the cognate codon-anticodon helix, initiating step-wise 'latching' of the decoding centre. The resulting closure of the 30S subunit docks EF-Tu at the sarcin-ricin loop of the 50S subunit, activating EF-Tu for GTP hydrolysis and enabling accommodation of the aminoacyl-tRNA. By contrast, near-cognate complexes fail to induce the G530 latch, thus favouring open 30S pre-accommodation intermediates with inactive EF-Tu. This work reveals long-sought structural differences between the pre-accommodation of cognate and near-cognate tRNAs that elucidate the mechanism of accurate decoding.


Assuntos
Microscopia Crioeletrônica , Biossíntese de Proteínas , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Anticódon/química , Anticódon/genética , Anticódon/ultraestrutura , Códon/química , Códon/genética , Códon/ultraestrutura , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/ultraestrutura , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/ultraestrutura , Guanosina Trifosfato/metabolismo , Hidrólise , Modelos Moleculares , Fator Tu de Elongação de Peptídeos/metabolismo , Fator Tu de Elongação de Peptídeos/ultraestrutura , Domínios Proteicos , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , RNA Ribossômico 16S/ultraestrutura , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/metabolismo , Aminoacil-RNA de Transferência/ultraestrutura , Subunidades Ribossômicas/química , Subunidades Ribossômicas/metabolismo , Subunidades Ribossômicas/ultraestrutura , Ribossomos/química
9.
Elife ; 52016 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-27434674

RESUMO

Stringent response is a conserved bacterial stress response underlying virulence and antibiotic resistance. RelA/SpoT-homolog proteins synthesize transcriptional modulators (p)ppGpp, allowing bacteria to adapt to stress. RelA is activated during amino-acid starvation, when cognate deacyl-tRNA binds to the ribosomal A (aminoacyl-tRNA) site. We report four cryo-EM structures of E. coli RelA bound to the 70S ribosome, in the absence and presence of deacyl-tRNA accommodating in the 30S A site. The boomerang-shaped RelA with a wingspan of more than 100 Å wraps around the A/R (30S A-site/RelA-bound) tRNA. The CCA end of the A/R tRNA pins the central TGS domain against the 30S subunit, presenting the (p)ppGpp-synthetase domain near the 30S spur. The ribosome and A/R tRNA are captured in three conformations, revealing hitherto elusive states of tRNA engagement with the ribosomal decoding center. Decoding-center rearrangements are coupled with the step-wise 30S-subunit 'closure', providing insights into the dynamics of high-fidelity tRNA decoding.


Assuntos
Escherichia coli/fisiologia , Ligases/metabolismo , Ligases/ultraestrutura , RNA de Transferência/metabolismo , RNA de Transferência/ultraestrutura , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Microscopia Crioeletrônica , Ligação Proteica , Estresse Fisiológico
10.
Nature ; 492(7428): 205-9, 2012 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-23201686

RESUMO

Replicative DNA helicases generally unwind DNA as a single hexamer that encircles and translocates along one strand of the duplex while excluding the complementary strand (known as steric exclusion). By contrast, large T antigen, the replicative DNA helicase of the simian virus 40 (SV40), is reported to function as a pair of stacked hexamers that pumps double-stranded DNA through its central channel while laterally extruding single-stranded DNA. Here we use single-molecule and ensemble assays to show that large T antigen assembled on the SV40 origin unwinds DNA efficiently as a single hexamer that translocates on single-stranded DNA in the 3'-to-5' direction. Unexpectedly, large T antigen unwinds DNA past a DNA-protein crosslink on the translocation strand, suggesting that the large T antigen ring can open to bypass bulky adducts. Together, our data underscore the profound conservation among replicative helicase mechanisms, and reveal a new level of plasticity in the interactions of replicative helicases with DNA damage.


Assuntos
DNA Helicases/metabolismo , Vírus 40 dos Símios/enzimologia , Antígenos Virais de Tumores/metabolismo , Replicação do DNA , DNA de Cadeia Simples/metabolismo , DNA Viral/metabolismo , Origem de Replicação/fisiologia , Proteínas Virais/metabolismo
11.
Nat Methods ; 9(10): 987-92, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22961247

RESUMO

Single-molecule fluorescence imaging is often incompatible with physiological protein concentrations, as fluorescence background overwhelms an individual molecule's signal. We solve this problem with a new imaging approach called PhADE (PhotoActivation, Diffusion and Excitation). A protein of interest is fused to a photoactivatable protein (mKikGR) and introduced to its surface-immobilized substrate. After photoactivation of mKikGR near the surface, rapid diffusion of the unbound mKikGR fusion out of the detection volume eliminates background fluorescence, whereupon the bound molecules are imaged. We labeled the eukaryotic DNA replication protein flap endonuclease 1 with mKikGR and added it to replication-competent Xenopus laevis egg extracts. PhADE imaging of high concentrations of the fusion construct revealed its dynamics and micrometer-scale movements on individual, replicating DNA molecules. Because PhADE imaging is in principle compatible with any photoactivatable fluorophore, it should have broad applicability in revealing single-molecule dynamics and stoichiometry of macromolecular protein complexes at previously inaccessible fluorophore concentrations.


Assuntos
Endonucleases Flap/química , Proteínas Luminescentes/química , Microscopia de Fluorescência/métodos , Replicação do DNA , Difusão , Antígeno Nuclear de Célula em Proliferação/química
12.
Methods ; 57(2): 179-86, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22503776

RESUMO

The recent advent in single-molecule imaging and manipulation methods has made a significant impact on the understanding of molecular mechanisms underlying many essential cellular processes. Single-molecule techniques such as electron microscopy and DNA fiber assays have been employed to study the duplication of genome in eukaryotes. Here, we describe a single-molecule assay that allows replication of DNA attached to the functionalized surface of a microfluidic flow cell in a soluble Xenopus leavis egg extract replication system and subsequent visualization of replication products via fluorescence microscopy. We also explain a method for detection of replication proteins, through fluorescently labeled antibodies, on partially replicated DNA immobilized at both ends to the surface.


Assuntos
Extratos Celulares/genética , Replicação do DNA , DNA Viral/biossíntese , Ácidos Nucleicos Imobilizados/biossíntese , Oócitos/citologia , Animais , Sequência de Bases , Separação Celular , DNA Viral/química , Feminino , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Ácidos Nucleicos Imobilizados/química , Técnicas Analíticas Microfluídicas/instrumentação , Antígeno Nuclear de Célula em Proliferação/química , Ligação Proteica , Coloração e Rotulagem , Xenopus laevis
13.
Mol Cell ; 40(5): 834-40, 2010 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-21145490

RESUMO

The duplication of eukaryotic genomes involves the replication of DNA from multiple origins of replication. In S phase, two sister replisomes assemble at each active origin, and they replicate DNA in opposite directions. Little is known about the functional relationship between sister replisomes. Some data imply that they travel away from one another and thus function independently. Alternatively, sister replisomes may form a stationary, functional unit that draws parental DNA toward itself. If this "double replisome" model is correct, a constrained DNA molecule should not undergo replication. To test this prediction, lambda DNA was stretched and immobilized at both ends within a microfluidic flow cell. Upon exposure to Xenopus egg extracts, this DNA underwent extensive replication by a single pair of diverging replisomes. The data show that there is no obligatory coupling between sister replisomes and, together with other studies, imply that genome duplication involves autonomously functioning replisomes.


Assuntos
Replicação do DNA , Animais , Genoma , Origem de Replicação , Fase S , Xenopus
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