RESUMEN
Ribosome rescue pathways recycle stalled ribosomes and target problematic mRNAs and aborted proteins for degradation1,2. In bacteria, it remains unclear how rescue pathways distinguish ribosomes stalled in the middle of a transcript from actively translating ribosomes3-6. Here, using a genetic screen in Escherichia coli, we discovered a new rescue factor that has endonuclease activity. SmrB cleaves mRNAs upstream of stalled ribosomes, allowing the ribosome rescue factor tmRNA (which acts on truncated mRNAs3) to rescue upstream ribosomes. SmrB is recruited to ribosomes and is activated by collisions. Cryo-electron microscopy structures of collided disomes from E. coli and Bacillus subtilis show distinct and conserved arrangements of individual ribosomes and the composite SmrB-binding site. These findings reveal the underlying mechanisms by which ribosome collisions trigger ribosome rescue in bacteria.
Asunto(s)
Escherichia coli , Ribosomas , Bacterias/genética , Microscopía por Crioelectrón , Escherichia coli/genética , Escherichia coli/metabolismo , Biosíntesis de Proteínas , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , Ribosomas/metabolismoRESUMEN
The stringent response enables bacteria to respond to nutrient limitation and other stress conditions through production of the nucleotide-based second messengers ppGpp and pppGpp, collectively known as (p)ppGpp. Here, we report that (p)ppGpp inhibits the signal recognition particle (SRP)-dependent protein targeting pathway, which is essential for membrane protein biogenesis and protein secretion. More specifically, (p)ppGpp binds to the SRP GTPases Ffh and FtsY, and inhibits the formation of the SRP receptor-targeting complex, which is central for the coordinated binding of the translating ribosome to the SecYEG translocon. Cryo-EM analysis of SRP bound to translating ribosomes suggests that (p)ppGpp may induce a distinct conformational stabilization of the NG domain of Ffh and FtsY in Bacillus subtilis but not in E. coli.
Asunto(s)
Proteínas de Escherichia coli , Partícula de Reconocimiento de Señal , Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Guanosina Pentafosfato/metabolismo , Unión Proteica , Receptores Citoplasmáticos y Nucleares/metabolismo , Partícula de Reconocimiento de Señal/metabolismoRESUMEN
In eukaryotic translation, termination and ribosome recycling phases are linked to subsequent initiation of a new round of translation by persistence of several factors at ribosomal sub-complexes. These comprise/include the large eIF3 complex, eIF3j (Hcr1 in yeast) and the ATP-binding cassette protein ABCE1 (Rli1 in yeast). The ATPase is mainly active as a recycling factor, but it can remain bound to the dissociated 40S subunit until formation of the next 43S pre-initiation complexes. However, its functional role and native architectural context remains largely enigmatic. Here, we present an architectural inventory of native yeast and human ABCE1-containing pre-initiation complexes by cryo-EM. We found that ABCE1 was mostly associated with early 43S, but also with later 48S phases of initiation. It adopted a novel hybrid conformation of its nucleotide-binding domains, while interacting with the N-terminus of eIF3j. Further, eIF3j occupied the mRNA entry channel via its ultimate C-terminus providing a structural explanation for its antagonistic role with respect to mRNA binding. Overall, the native human samples provide a near-complete molecular picture of the architecture and sophisticated interaction network of the 43S-bound eIF3 complex and the eIF2 ternary complex containing the initiator tRNA.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Línea Celular , Proteínas de Unión al ADN/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Células HEK293 , Humanos , Unión Proteica/fisiología , Biosíntesis de Proteínas/fisiología , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Bacterial flagella differ in their number and spatial arrangement. In many species, the MinD-type ATPase FlhG (also YlxH/FleN) is central to the numerical control of bacterial flagella, and its deletion in polarly flagellated bacteria typically leads to hyperflagellation. The molecular mechanism underlying this numerical control, however, remains enigmatic. Using the model species Shewanella putrefaciens, we show that FlhG links assembly of the flagellar C ring with the action of the master transcriptional regulator FlrA (named FleQ in other species). While FlrA and the flagellar C-ring protein FliM have an overlapping binding site on FlhG, their binding depends on the ATP-dependent dimerization state of FlhG. FliM interacts with FlhG independent of nucleotide binding, while FlrA exclusively interacts with the ATP-dependent FlhG dimer and stimulates FlhG ATPase activity. Our in vivo analysis of FlhG partner switching between FliM and FlrA reveals its mechanism in the numerical restriction of flagella, in which the transcriptional activity of FlrA is down-regulated through a negative feedback loop. Our study demonstrates another level of regulatory complexity underlying the spationumerical regulation of flagellar biogenesis and implies that flagellar assembly transcriptionally regulates the production of more initial building blocks.
Asunto(s)
Proteínas Bacterianas/metabolismo , Flagelos/genética , Flagelos/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Bacterias/metabolismo , Fenómenos Bioquímicos , Expresión Génica/genética , Regulación Bacteriana de la Expresión Génica/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Shewanella putrefaciens/genética , Shewanella putrefaciens/metabolismoRESUMEN
Cells adjust to nutrient deprivation by reversible translational shutdown. This is accompanied by maintaining inactive ribosomes in a hibernation state, in which they are bound by proteins with inhibitory and protective functions. In eukaryotes, such a function was attributed to suppressor of target of Myb protein 1 (Stm1; SERPINE1 mRNA-binding protein 1 [SERBP1] in mammals), and recently, late-annotated short open reading frame 2 (Lso2; coiled-coil domain containing short open reading frame 124 [CCDC124] in mammals) was found to be involved in translational recovery after starvation from stationary phase. Here, we present cryo-electron microscopy (cryo-EM) structures of translationally inactive yeast and human ribosomes. We found Lso2/CCDC124 accumulating on idle ribosomes in the nonrotated state, in contrast to Stm1/SERBP1-bound ribosomes, which display a rotated state. Lso2/CCDC124 bridges the decoding sites of the small with the GTPase activating center (GAC) of the large subunit. This position allows accommodation of the duplication of multilocus region 34 protein (Dom34)-dependent ribosome recycling system, which splits Lso2-containing, but not Stm1-containing, ribosomes. We propose a model in which Lso2 facilitates rapid translation reactivation by stabilizing the recycling-competent state of inactive ribosomes.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada , Evolución Molecular , Células HEK293 , Humanos , Modelos Moleculares , Péptidos/química , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribosomas/ultraestructura , Saccharomyces cerevisiae/ultraestructura , Relación Estructura-ActividadRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. A major virulence factor of SARS-CoVs is the nonstructural protein 1 (Nsp1), which suppresses host gene expression by ribosome association. Here, we show that Nsp1 from SARS-CoV-2 binds to the 40S ribosomal subunit, resulting in shutdown of messenger RNA (mRNA) translation both in vitro and in cells. Structural analysis by cryo-electron microscopy of in vitro-reconstituted Nsp1-40S and various native Nsp1-40S and -80S complexes revealed that the Nsp1 C terminus binds to and obstructs the mRNA entry tunnel. Thereby, Nsp1 effectively blocks retinoic acid-inducible gene I-dependent innate immune responses that would otherwise facilitate clearance of the infection. Thus, the structural characterization of the inhibitory mechanism of Nsp1 may aid structure-based drug design against SARS-CoV-2.
Asunto(s)
Betacoronavirus/química , Evasión Inmune , Inmunidad Innata , Biosíntesis de Proteínas , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Betacoronavirus/inmunología , Betacoronavirus/metabolismo , Betacoronavirus/fisiología , Sitios de Unión , COVID-19 , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Microscopía por Crioelectrón , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/metabolismo , Humanos , Interferón beta/genética , Interferón beta/metabolismo , Modelos Moleculares , Pandemias , Neumonía Viral/inmunología , Neumonía Viral/virología , Unión Proteica , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , ARN Mensajero/metabolismo , Receptores Inmunológicos , Subunidades Ribosómicas Pequeñas de Eucariotas/química , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , SARS-CoV-2RESUMEN
Ribosome recycling by the twin-ATPase ABCE1 is a key regulatory process in mRNA translation and surveillance and in ribosome-associated protein quality control in Eukarya and Archaea. Here, we captured the archaeal 30S ribosome post-splitting complex at 2.8 Å resolution by cryo-electron microscopy. The structure reveals the dynamic behavior of structural motifs unique to ABCE1, which ultimately leads to ribosome splitting. More specifically, we provide molecular details on how conformational rearrangements of the iron-sulfur cluster domain and hinge regions of ABCE1 are linked to closure of its nucleotide-binding sites. The combination of mutational and functional analyses uncovers an intricate allosteric network between the ribosome, regulatory domains of ABCE1, and its two structurally and functionally asymmetric ATP-binding sites. Based on these data, we propose a refined model of how signals from the ribosome are integrated into the ATPase cycle of ABCE1 to orchestrate ribosome recycling.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Subunidades Ribosómicas Pequeñas de Archaea/metabolismo , Thermococcus/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Microscopía por Crioelectrón , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Subunidades Ribosómicas Pequeñas de Archaea/química , Ribosomas/metabolismo , Thermococcus/genéticaRESUMEN
Chemical modifications of human ribosomal RNA (rRNA) are introduced during biogenesis and have been implicated in the dysregulation of protein synthesis, as is found in cancer and other diseases. However, their role in this phenomenon is unknown. Here we visualize more than 130 individual rRNA modifications in the three-dimensional structure of the human ribosome, explaining their structural and functional roles. In addition to a small number of universally conserved sites, we identify many eukaryote- or human-specific modifications and unique sites that form an extended shell in comparison to bacterial ribosomes, and which stabilize the RNA. Several of the modifications are associated with the binding sites of three ribosome-targeting antibiotics, or are associated with degenerate states in cancer, such as keto alkylations on nucleotide bases reminiscent of specialized ribosomes. This high-resolution structure of the human 80S ribosome paves the way towards understanding the role of epigenetic rRNA modifications in human diseases and suggests new possibilities for designing selective inhibitors and therapeutic drugs.
Asunto(s)
Microscopía por Crioelectrón , ARN Ribosómico/química , ARN Ribosómico/ultraestructura , Ribosomas/química , Ribosomas/ultraestructura , Sitios de Unión , Epistasis Genética , Células HeLa , Humanos , Ligandos , Modelos Moleculares , Estabilidad del ARN , ARN Ribosómico/biosíntesis , ARN Ribosómico/clasificación , Subunidades Ribosómicas Grandes de Eucariotas/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Ribosomas/efectos de los fármacos , Ribosomas/genéticaRESUMEN
Cryo electron microscopy (cryo-EM) historically has had a strong impact on the structural and mechanistic analysis of protein synthesis by the prokaryotic and eukaryotic ribosomes. Vice versa, studying ribosomes has helped moving forwards many methodological aspects in single particle cryo-EM, at the level of automated data collection and image processing including advanced techniques for particle sorting to address structural and compositional heterogeneity. Here we review some of the latest ribosome structures, where cryo-EM allowed gaining unprecedented insights based on 3D structure sorting with focused classification and refinement methods helping to reach local resolution levels better than 3Å. Such high-resolution features now enable the analysis of drug interactions with RNA and protein side-chains including even the visualization of chemical modifications of the ribosomal RNA. These advances represent a major breakthrough in structural biology and show the strong potential of cryo-EM beyond the ribosome field including for structure-based drug design.
Asunto(s)
Microscopía por Crioelectrón/métodos , Ribosomas/química , ARN Ribosómico/química , ARN Ribosómico/metabolismo , Ribosomas/metabolismoRESUMEN
After gradually moving away from preparation methods prone to artefacts such as plastic embedding and negative staining for cell sections and single particles, the field of cryo electron microscopy (cryo-EM) is now heading off at unprecedented speed towards high-resolution analysis of biological objects of various sizes. This 'revolution in resolution' is happening largely thanks to new developments of new-generation cameras used for recording the images in the cryo electron microscope which have much increased sensitivity being based on complementary metal oxide semiconductor devices. Combined with advanced image processing and 3D reconstruction, the cryo-EM analysis of nucleoprotein complexes can provide unprecedented insights at molecular and atomic levels and address regulatory mechanisms in the cell. These advances reinforce the integrative role of cryo-EM in synergy with other methods such as X-ray crystallography, fluorescence imaging or focussed-ion beam milling as exemplified here by some recent studies from our laboratory on ribosomes, viruses, chromatin and nuclear receptors. Such multi-scale and multi-resolution approaches allow integrating molecular and cellular levels when applied to purified or in situ macromolecular complexes, thus illustrating the trend of the field towards cellular structural biology.
Asunto(s)
Microscopía por Crioelectrón , Animales , Cristalografía por Rayos X , Humanos , Sustancias Macromoleculares/ultraestructura , Modelos Moleculares , Conformación Molecular , Imagen Individual de Molécula , TomografíaRESUMEN
Flavonoids represent a large class of secondary metabolites produced by plants. These polyphenolic compounds are well known for their antioxidative abilities, are antimicrobial phytoalexins responsible for flower pigmentation to attract pollinators and, in addition to other properties, are also specific bacterial regulators governing the expression of Rhizobium genes involved in root nodulation (Firmin et al., 1986). The bacterial chalcone isomerase (CHI) from Eubacterium ramulus catalyses the first step in a flavanone-degradation pathway by ring opening of (2S)-naringenin to form naringenin chalcone. The structural biology and enzymology of plant CHIs have been well documented, whereas the existence of bacterial CHIs has only recently been elucidated. This first determination of the structure of a bacterial CHI provides detailed structural insights into the key step of the flavonoid-degradation pathway. The active site could be confirmed by co-crystallization with the substrate (2S)-naringenin. The stereochemistry of the proposed mechanism of the isomerase reaction was verified by specific (1)H/(2)H isotope exchange observed by (1)H NMR experiments and was further supported by mutagenesis studies. The active site is shielded by a flexible lid, the varying structure of which could be modelled in different states of the catalytic cycle using small-angle X-ray scattering data together with the crystallographic structures. Comparison of bacterial CHI with the plant enzyme from Medicago sativa reveals that they have unrelated folds, suggesting that the enzyme activity evolved convergently from different ancestor proteins. Despite the lack of any functional relationship, the tertiary structure of the bacterial CHI shows similarities to the ferredoxin-like fold of a chlorite dismutase and the stress-related protein SP1.