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1.
Elife ; 82019 Sep 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.

3.
Am J Hum Genet ; 2018 Nov 21.
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.

4.
J Biol Chem ; 293(32): 12472-12479, 2018 Aug 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.

5.
Structure ; 26(6): 821-828.e3, 2018 Jun 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.

6.
J Mol Biol ; 430(5): 591-593, 2018 Mar 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.

7.
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.

8.
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
9.
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
10.
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
11.
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
12.
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
13.
Elife ; 52016 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-27159452

RESUMO

Internal ribosome entry sites (IRESs) mediate cap-independent translation of viral mRNAs. Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2•GTP bound with sordarin. The structures suggest a trajectory of IRES translocation, required for translation initiation, and provide an unprecedented view of eEF2 dynamics. The IRES rearranges from extended to bent to extended conformations. This inchworm-like movement is coupled with ribosomal inter-subunit rotation and 40S head swivel. eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site. Its diphthamide-bearing tip at domain IV separates the tRNA-mRNA-like pseudoknot I (PKI) of the IRES from the decoding center. This unlocks 40S domains, facilitating head swivel and biasing IRES translocation via hitherto-elusive intermediates with PKI captured between the A and P sites. The structures suggest missing links in our understanding of tRNA translocation.


Assuntos
Sítios Internos de Entrada Ribossomal , Fator 2 de Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Microscopia Crioeletrônica/métodos , Dicistroviridae/genética , Guanosina Trifosfato/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Substâncias Macromoleculares/metabolismo , Substâncias Macromoleculares/ultraestrutura , Conformação de Ácido Nucleico , Fator 2 de Elongação de Peptídeos/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/ultraestrutura , RNA Viral/genética , RNA Viral/ultraestrutura , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Saccharomyces cerevisiae/genética
14.
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
15.
Nucleic Acids Res ; 44(1): 95-105, 2016 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-26673695

RESUMO

Easy-to-use macromolecular viewers, such as UCSF Chimera, are a standard tool in structural biology. They allow rendering and performing geometric operations on large complexes, such as viruses and ribosomes. Dynamical simulation codes enable modeling of conformational changes, but may require considerable time and many CPUs. There is an unmet demand from structural and molecular biologists for software in the middle ground, which would allow visualization combined with quick and interactive modeling of conformational changes, even of large complexes. This motivates MMB-GUI. MMB uses an internal-coordinate, multiscale approach, yielding as much as a 2000-fold speedup over conventional simulation methods. We use Chimera as an interactive graphical interface to control MMB. We show how this can be used for morphing of macromolecules that can be heterogeneous in biopolymer type, sequence, and chain count, accurately recapitulating structural intermediates. We use MMB-GUI to create a possible trajectory of EF-G mediated gate-passing translocation in the ribosome, with all-atom structures. This shows that the GUI makes modeling of large macromolecules accessible to a wide audience. The morph highlights similarities in tRNA conformational changes as tRNA translocates from A to P and from P to E sites and suggests that tRNA flexibility is critical for translocation completion.


Assuntos
RNA de Transferência/química , RNA de Transferência/genética , Ribossomos/química , Ribossomos/metabolismo , Interface Usuário-Computador , Modelos Moleculares , Conformação Molecular , Fator G para Elongação de Peptídeos/química , Fator G para Elongação de Peptídeos/metabolismo , Ligação Proteica
16.
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
17.
Nature ; 519(7541): 110-3, 2015 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-25652826

RESUMO

The central dogma of gene expression (DNA to RNA to protein) is universal, but in different domains of life there are fundamental mechanistic differences within this pathway. For example, the canonical molecular signals used to initiate protein synthesis in bacteria and eukaryotes are mutually exclusive. However, the core structures and conformational dynamics of ribosomes that are responsible for the translation steps that take place after initiation are ancient and conserved across the domains of life. We wanted to explore whether an undiscovered RNA-based signal might be able to use these conserved features, bypassing mechanisms specific to each domain of life, and initiate protein synthesis in both bacteria and eukaryotes. Although structured internal ribosome entry site (IRES) RNAs can manipulate ribosomes to initiate translation in eukaryotic cells, an analogous RNA structure-based mechanism has not been observed in bacteria. Here we report our discovery that a eukaryotic viral IRES can initiate translation in live bacteria. We solved the crystal structure of this IRES bound to a bacterial ribosome to 3.8 Å resolution, revealing that despite differences between bacterial and eukaryotic ribosomes this IRES binds directly to both and occupies the space normally used by transfer RNAs. Initiation in both bacteria and eukaryotes depends on the structure of the IRES RNA, but in bacteria this RNA uses a different mechanism that includes a form of ribosome repositioning after initial recruitment. This IRES RNA bridges billions of years of evolutionary divergence and provides an example of an RNA structure-based translation initiation signal capable of operating in two domains of life.


Assuntos
Bactérias/genética , Eucariotos/genética , Conformação de Ácido Nucleico , Biossíntese de Proteínas/genética , RNA/química , RNA/genética , Ribossomos/metabolismo , Sequência de Bases , Sequência Conservada/genética , Cristalografia por Raios X , Dicistroviridae/genética , Modelos Moleculares , Iniciação Traducional da Cadeia Peptídica/genética , RNA/metabolismo , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Ribossomos/química
18.
Cell ; 159(3): 475-6, 2014 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-25417100

RESUMO

Eukaryotic translation initiation requires coordinated assembly of a remarkable array of initiation factors onto the small ribosomal subunit to select an appropriate mRNA start codon. Studies from Erzberger et al. and Hussain et al. bring new insights into this mechanism by looking at early and late initiation intermediates.


Assuntos
Fator de Iniciação 1 em Eucariotos/química , Fator de Iniciação 3 em Eucariotos/química , Fatores de Iniciação em Eucariotos/metabolismo , Kluyveromyces/metabolismo , Iniciação Traducional da Cadeia Peptídica , Subunidades Ribossômicas Menores de Eucariotos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Animais , Humanos
19.
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
20.
Proc Natl Acad Sci U S A ; 111(25): 9139-44, 2014 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-24927574

RESUMO

In cap-dependent translation initiation, the open reading frame (ORF) of mRNA is established by the placement of the AUG start codon and initiator tRNA in the ribosomal peptidyl (P) site. Internal ribosome entry sites (IRESs) promote translation of mRNAs in a cap-independent manner. We report two structures of the ribosome-bound Taura syndrome virus (TSV) IRES belonging to the family of Dicistroviridae intergenic IRESs. Intersubunit rotational states differ in these structures, suggesting that ribosome dynamics play a role in IRES translocation. Pseudoknot I of the IRES occupies the ribosomal decoding center at the aminoacyl (A) site in a manner resembling that of the tRNA anticodon-mRNA codon. The structures reveal that the TSV IRES initiates translation by a previously unseen mechanism, which is conceptually distinct from initiator tRNA-dependent mechanisms. Specifically, the ORF of the IRES-driven mRNA is established by the placement of the preceding tRNA-mRNA-like structure in the A site, whereas the 40S P site remains unoccupied during this initial step.


Assuntos
Conformação de Ácido Nucleico , Iniciação Traducional da Cadeia Peptídica , Picornaviridae/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , RNA Viral/metabolismo , Ribossomos/metabolismo , Fases de Leitura Aberta , Picornaviridae/genética , RNA Mensageiro/genética , RNA de Transferência/genética , RNA Viral/genética , Ribossomos/genética
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