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1.
Biochemistry (Mosc) ; 86(9): 1107-1121, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34565314

RESUMO

When a ribosome encounters the stop codon of an mRNA, it terminates translation, releases the newly made protein, and is recycled to initiate translation on a new mRNA. Termination is a highly dynamic process in which release factors (RF1 and RF2 in bacteria; eRF1•eRF3•GTP in eukaryotes) coordinate peptide release with large-scale molecular rearrangements of the ribosome. Ribosomes stalled on aberrant mRNAs are rescued and recycled by diverse bacterial, mitochondrial, or cytoplasmic quality control mechanisms. These are catalyzed by rescue factors with peptidyl-tRNA hydrolase activity (bacterial ArfA•RF2 and ArfB, mitochondrial ICT1 and mtRF-R, and cytoplasmic Vms1), that are distinct from each other and from release factors. Nevertheless, recent structural studies demonstrate a remarkable similarity between translation termination and ribosome rescue mechanisms. This review describes how these pathways rely on inherent ribosome dynamics, emphasizing the active role of the ribosome in all translation steps.


Assuntos
Bactérias/metabolismo , Citoplasma/metabolismo , Mitocôndrias/metabolismo , Ribossomos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Terminação Traducional da Cadeia Peptídica , Biossíntese de Proteínas , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo
2.
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
3.
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
4.
RNA ; 26(12): 2044-2050, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32817446

RESUMO

Termination of protein biosynthesis is an essential step of gene expression, during which a complete functional protein is released from the ribosome. Premature or inefficient termination results in truncated, nonfunctional, or toxic proteins that may cause disease. Indeed, more than 10% of human genetic diseases are caused by nonsense mutations leading to premature termination. Efficient and sensitive approaches are required to study eukaryotic termination mechanisms and to identify potential therapeutics that modulate termination. Canonical radioactivity-based termination assays are complex, report on a short peptide release, and are incompatible with high-throughput screening. Here we describe a robust and simple in vitro assay to study the kinetics of full-protein release. The assay monitors luminescence upon release of nanoluciferase from a mammalian pretermination complex. The assay can be used to record time-progress curves of protein release in a high-throughput format, making it optimal for studying release kinetics and for high-throughput screening for small molecules that modulate the efficiency of termination.


Assuntos
Bioensaio/métodos , Luciferases/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Humanos , Terminação Traducional da Cadeia Peptídica
5.
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
6.
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
7.
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
8.
Elife ; 92020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32427100

RESUMO

Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Förster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape the ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation.


Assuntos
Conformação de Ácido Nucleico , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Ribossomos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Microscopia Crioeletrônica , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Mudança da Fase de Leitura do Gene Ribossômico , Proteínas de Fusão gag-pol , Regulação Bacteriana da Expressão Gênica , Regulação Viral da Expressão Gênica , HIV-1/genética , HIV-1/metabolismo , RNA Bacteriano , RNA Mensageiro/química , RNA de Transferência/química
9.
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
11.
Am J Hum Genet ; 103(6): 930-947, 2018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30503522

RESUMO

Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 7 out of 1,000,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole-exome sequencing (WES). We identified relevant rare and predicted damaging mutations for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and located in 1 of 19 previously reported ribosomal protein (RP)-encoding genes. Using exon coverage estimates, we identified and validated 31 deletions in RP genes. We also observed an enrichment for extended splice site mutations and validated their diverse effects using RNA sequencing in cell lines obtained from individuals with DBA. Leveraging the size of our cohort, we observed robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. We further identified rare mutations in seven previously unreported RP genes that may cause DBA, as well as several distinct disorders that appear to phenocopy DBA, including nine individuals with biallelic CECR1 mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain >5% of DBA-affected case subjects. Overall, this report should inform not only clinical practice for DBA-affected individuals, but also the design and analysis of rare variant studies for heterogeneous Mendelian disorders.


Assuntos
Anemia de Diamond-Blackfan/genética , Adolescente , Criança , Pré-Escolar , Estudos de Coortes , Exoma/genética , Éxons/genética , Feminino , Deleção de Genes , Estudos de Associação Genética/métodos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Mutação/genética , Fenótipo , Proteínas Ribossômicas/genética , Ribossomos/genética , Análise de Sequência de RNA/métodos , Sequenciamento Completo do Exoma/métodos
12.
J Biol Chem ; 293(32): 12472-12479, 2018 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-29941456

RESUMO

Accurate translation termination by release factors (RFs) is critical for the integrity of cellular proteomes. Premature termination on sense codons, for example, results in truncated proteins, whose accumulation could be detrimental to the cell. Nevertheless, some sense codons are prone to triggering premature termination, but the structural basis for this is unclear. To investigate premature termination, we determined a cryo-EM structure of the Escherichia coli 70S ribosome bound with RF1 in response to a UAU (Tyr) sense codon. The structure reveals that RF1 recognizes a UAU codon similarly to a UAG stop codon, suggesting that sense codons induce premature termination because they structurally mimic a stop codon. Hydrophobic interaction between the nucleobase of U3 (the third position of the UAU codon) and conserved Ile-196 in RF1 is important for misreading the UAU codon. Analyses of RNA binding in ribonucleoprotein complexes or by amino acids reveal that Ile-U packing is a frequent protein-RNA-binding motif with key functional implications. We discuss parallels with eukaryotic translation termination by the release factor eRF1.


Assuntos
Códon de Terminação/metabolismo , Códon/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/metabolismo , Ribossomos/metabolismo , Códon/química , Códon/genética , Códon de Terminação/química , Códon de Terminação/genética , Cristalografia por Raios X , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/genética , Conformação Proteica , Ribossomos/química
13.
Structure ; 26(6): 821-828.e3, 2018 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-29731232

RESUMO

Translation termination ensures proper lengths of cellular proteins. During termination, release factor (RF) recognizes a stop codon and catalyzes peptide release. Conformational changes in RF are thought to underlie accurate translation termination. However, structural studies of ribosome termination complexes have only captured RFs in a conformation that is consistent with the catalytically active state. Here, we employ a hyper-accurate RF1 variant to obtain crystal structures of 70S termination complexes that suggest a structural pathway for RF1 activation. We trapped RF1 conformations with the catalytic domain outside of the peptidyl-transferase center, while the codon-recognition domain binds the stop codon. Stop-codon recognition induces 30S decoding-center rearrangements that precede accommodation of the catalytic domain. The separation of codon recognition from the opening of the catalytic domain suggests how rearrangements in RF1 and in the ribosomal decoding center coordinate stop-codon recognition with peptide release, ensuring accurate translation termination.


Assuntos
Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Sítios de Ligação , Domínio Catalítico , Códon de Terminação , Modelos Moleculares , Terminação Traducional da Cadeia Peptídica , Ligação Proteica , Conformação Proteica , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/metabolismo
14.
J Mol Biol ; 430(5): 591-593, 2018 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-29366636

RESUMO

Understanding the mechanisms of inhibitors of translation termination may inform development of new antibacterials and therapeutics for premature termination diseases. We report the crystal structure of the potent termination inhibitor blasticidin S bound to the ribosomal 70S•release factor 1 (RF1) termination complex. Blasticidin S shifts the catalytic domain 3 of RF1 and restructures the peptidyl transferase center. Universally conserved uridine 2585 in the peptidyl transferase center occludes the catalytic backbone of the GGQ motif of RF1, explaining the structural mechanism of inhibition. Rearrangement of domain 3 relative to the codon-recognition domain 2 provides insight into the dynamics of RF1 implicated in termination accuracy.


Assuntos
Antibacterianos/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Domínio Catalítico/efeitos dos fármacos , Códon de Terminação/metabolismo , Modelos Moleculares , Nucleosídeos/antagonistas & inibidores , Terminação Traducional da Cadeia Peptídica/efeitos dos fármacos , Fatores de Terminação de Peptídeos/efeitos dos fármacos , Peptidil Transferases/metabolismo , Conformação Proteica , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo
15.
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
16.
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
17.
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
18.
Sci Rep ; 7(1): 969, 2017 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-28428565

RESUMO

Eubacterial ribosomal large-subunit methyltransferase H (RlmH) methylates 23S ribosomal RNA pseudouridine 1915 (Ψ1915), which lies near the ribosomal decoding center. The smallest member of the SPOUT superfamily of methyltransferases, RlmH lacks the RNA recognition domain found in larger methyltransferases. The catalytic mechanism of RlmH enzyme is unknown. Here, we describe the structures of RlmH bound to S-adenosyl-methionine (SAM) and the methyltransferase inhibitor sinefungin. Our structural and biochemical studies reveal catalytically essential residues in the dimer-mediated asymmetrical active site. One monomer provides the SAM-binding site, whereas the conserved C-terminal tail of the second monomer provides residues essential for catalysis. Our findings elucidate the mechanism by which a small protein dimer assembles a functionally asymmetric architecture.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Metiltransferases/química , Metiltransferases/metabolismo , RNA Ribossômico 23S/química , S-Adenosilmetionina/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Sítios de Ligação , Domínio Catalítico , Escherichia coli/química , Proteínas de Escherichia coli/genética , Metilação , Metiltransferases/genética , Modelos Moleculares , Mutação , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Pseudouridina/metabolismo , Especificidade por Substrato
19.
Elife ; 62017 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-28300532

RESUMO

ArfA rescues ribosomes stalled on truncated mRNAs by recruiting release factor RF2, which normally binds stop codons to catalyze peptide release. We report two 3.2 Šresolution cryo-EM structures - determined from a single sample - of the 70S ribosome with ArfA•RF2 in the A site. In both states, the ArfA C-terminus occupies the mRNA tunnel downstream of the A site. One state contains a compact inactive RF2 conformation. Ordering of the ArfA N-terminus in the second state rearranges RF2 into an extended conformation that docks the catalytic GGQ motif into the peptidyl-transferase center. Our work thus reveals the structural dynamics of ribosome rescue. The structures demonstrate how ArfA 'senses' the vacant mRNA tunnel and activates RF2 to mediate peptide release without a stop codon, allowing stalled ribosomes to be recycled.


Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestrutura , Fatores de Terminação de Peptídeos/metabolismo , Fatores de Terminação de Peptídeos/ultraestrutura , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/ultraestrutura , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Microscopia Crioeletrônica , Ligação Proteica
20.
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
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