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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): 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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
J Mol Biol ; 428(10 Pt B): 2228-36, 2016 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-27107638

RESUMO

Pseudouridylation of messenger RNA emerges as an abundant modification involved in gene expression regulation. Pseudouridylation of stop codons in eukaryotic and bacterial cells results in stop-codon read through. The structural mechanism of this phenomenon is not known. Here we present a 3.1-Å crystal structure of Escherichia coli release factor 1 (RF1) bound to the 70S ribosome in response to the ΨAA codon. The structure reveals that recognition of a modified stop codon does not differ from that of a canonical stop codon. Our in vitro biochemical results support this finding by yielding nearly identical rates for peptide release from E. coli ribosomes programmed with pseudouridylated and canonical stop codons. The crystal structure also brings insight into E. coli RF1-specific interactions and suggests involvement of L27 in bacterial translation termination. Our results are consistent with a mechanism in which read through of a pseudouridylated stop codon in bacteria results from increased decoding by near-cognate tRNAs (miscoding) rather than from decreased efficiency of termination.


Assuntos
Códon de Terminação/genética , Terminação Traducional da Cadeia Peptídica/genética , Biossíntese de Proteínas/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fatores de Terminação de Peptídeos/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Ribossomos/genética
9.
Structure ; 23(11): 2155-61, 2015 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-26412335

RESUMO

Translation initiation in the P site occasionally occurs at atypical (non-AUG) start codons, including those forming a mismatch in the third (wobble) position. During elongation, however, a pyrimidine-pyrimidine wobble mismatch may trigger a translation quality-control mechanism, whereby the P-site mismatch is thought to perturb the downstream A-site codon or the decoding center, thereby reducing translation fidelity and inducing termination of aberrant translation. We report a crystal structure of the 70S initiation complex containing an AUC codon in the ribosomal P site. Remarkably, the ribosome stabilizes the mismatched codon-anticodon helix, arranging a normally disruptive cytosine-cytosine pair into a Watson-Crick-like conformation. Translation-competent conformations of the tRNA, mRNA, and decoding center suggest that a P-site wobble-position mismatch in the 70S initiation complex does not pre-arrange the mRNA or decoding center to favor subsequent miscoding events.


Assuntos
Pareamento de Bases , Domínio Catalítico , Códon de Iniciação/química , Ribossomos/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Dados de Sequência Molecular , Iniciação Traducional da Cadeia Peptídica , Ligação Proteica , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribossomos/metabolismo
10.
Structure ; 22(8): 1210-1218, 2014 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-25043550

RESUMO

The structural understanding of eukaryotic translation lags behind that of translation on bacterial ribosomes. Here, we present two subnanometer resolution structures of S. cerevisiae 80S ribosome complexes formed with either one or two tRNAs and bound in response to an mRNA fragment containing the Kozak consensus sequence. The ribosomes adopt two globally different conformations that are related to each other by the rotation of the small subunit. Comparison with bacterial ribosome complexes reveals that the global structures and modes of intersubunit rotation of the yeast ribosome differ significantly from those in the bacterial counterpart, most notably in the regions involving the tRNA, small ribosomal subunit, and conserved helix 69 of the large ribosomal subunit. The structures provide insight into ribosome dynamics implicated in tRNA translocation and help elucidate the role of the Kozak fragment in positioning an open reading frame during translation initiation in eukaryotes.


Assuntos
Modelos Moleculares , Conformação Molecular , RNA de Transferência/química , Ribossomos/química , Proteínas de Saccharomyces cerevisiae/química , Microscopia Crioeletrônica , Processamento de Imagem Assistida por Computador , Biossíntese de Proteínas/genética , RNA de Transferência/metabolismo , Ribossomos/metabolismo
11.
Proc Natl Acad Sci U S A ; 110(30): 12283-8, 2013 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-23824292

RESUMO

The antibiotic blasticidin S (BlaS) is a potent inhibitor of protein synthesis in bacteria and eukaryotes. We have determined a 3.4-Šcrystal structure of BlaS bound to a 70S⋅tRNA ribosome complex and performed biochemical and single-molecule FRET experiments to determine the mechanism of action of the antibiotic. We find that BlaS enhances tRNA binding to the P site of the large ribosomal subunit and slows down spontaneous intersubunit rotation in pretranslocation ribosomes. However, the antibiotic has negligible effect on elongation factor G catalyzed translocation of tRNA and mRNA. The crystal structure of the antibiotic-ribosome complex reveals that BlaS impedes protein synthesis through a unique mechanism by bending the 3' terminus of the P-site tRNA toward the A site of the large ribosomal subunit. Biochemical experiments demonstrate that stabilization of the deformed conformation of the P-site tRNA by BlaS strongly inhibits peptidyl-tRNA hydrolysis by release factors and, to a lesser extent, peptide bond formation.


Assuntos
Biossíntese de Proteínas/efeitos dos fármacos , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Nucleosídeos/farmacologia , RNA de Transferência/química , Thermus thermophilus/metabolismo
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