RESUMEN
Cellular proteins begin to fold as they emerge from the ribosome. The folding landscape of nascent chains is not only shaped by their amino acid sequence but also by the interactions with the ribosome. Here, we combine biophysical methods with cryo-EM structure determination to show that folding of a ß-barrel protein begins with formation of a dynamic α-helix inside the ribosome. As the growing peptide reaches the end of the tunnel, the N-terminal part of the nascent chain refolds to a ß-hairpin structure that remains dynamic until its release from the ribosome. Contacts with the ribosome and structure of the peptidyl transferase center depend on nascent chain conformation. These results indicate that proteins may start out as α-helices inside the tunnel and switch into their native folds only as they emerge from the ribosome. Moreover, the correlation of nascent chain conformations with reorientation of key residues of the ribosomal peptidyl-transferase center suggest that protein folding could modulate ribosome activity.
Asunto(s)
Proteínas y Péptidos de Choque por Frío/química , Proteínas y Péptidos de Choque por Frío/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Dicroismo Circular , Proteínas y Péptidos de Choque por Frío/genética , Microscopía por Crioelectrón , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Modelos Moleculares , Biosíntesis de Proteínas , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Pliegue de Proteína , Procesamiento Proteico-Postraduccional , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
Hummingbirds are very well adapted to sustain efficient and rapid metabolic shifts. They oxidize ingested nectar to directly fuel flight when foraging but have to switch to oxidizing stored lipids derived from ingested sugars during the night or long-distance migratory flights. Understanding how this organism moderates energy turnover is hampered by a lack of information regarding how relevant enzymes differ in sequence, expression, and regulation. To explore these questions, we generated a chromosome-scale genome assembly of the ruby-throated hummingbird (A. colubris) using a combination of long- and short-read sequencing, scaffolding it using existing assemblies. We then used hybrid long- and short-read RNA sequencing of liver and muscle tissue in fasted and fed metabolic states for a comprehensive transcriptome assembly and annotation. Our genomic and transcriptomic data found positive selection of key metabolic genes in nectivorous avian species and deletion of critical genes (SLC2A4, GCK) involved in glucostasis in other vertebrates. We found expression of a fructose-specific version of SLC2A5 putatively in place of insulin-sensitive SLC2A5, with predicted protein models suggesting affinity for both fructose and glucose. Alternative isoforms may even act to sequester fructose to preclude limitations from transport in metabolism. Finally, we identified differentially expressed genes from fasted and fed hummingbirds, suggesting key pathways for the rapid metabolic switch hummingbirds undergo.
Asunto(s)
Aves , Metabolismo Energético , Animales , Aves/genética , Músculos/metabolismo , Genómica , Fructosa/metabolismoRESUMEN
A crucial step in translation is the translocation of tRNAs through the ribosome. In the transition from one canonical site to the other, the tRNAs acquire intermediate configurations, so-called hybrid states. At this stage, the small subunit is rotated with respect to the large subunit, and the anticodon stem loops reside in the A and P sites of the small subunit, while the acceptor ends interact with the P and E sites of the large subunit. In this work, by means of cryo-EM and particle classification procedures, we visualize the hybrid state of both A/P and P/E tRNAs in an authentic factor-free ribosome complex during translocation. In addition, we show how the repositioning of the tRNAs goes hand in hand with the change in the interplay between S13, L1 stalk, L5, H68, H69, and H38 that is caused by the ratcheting of the small subunit.
Asunto(s)
ARN de Transferencia/ultraestructura , Subunidades Ribosómicas Grandes Bacterianas/ultraestructura , Subunidades Ribosómicas Pequeñas Bacterianas/ultraestructura , Sitios de Unión , Microscopía por Crioelectrón , Modelos Moleculares , Conformación de Ácido Nucleico , Extensión de la Cadena Peptídica de Translación , Biosíntesis de Proteínas , Subunidades de Proteína/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Subunidades Ribosómicas Grandes Bacterianas/química , Subunidades Ribosómicas Grandes Bacterianas/fisiología , Subunidades Ribosómicas Pequeñas Bacterianas/química , Subunidades Ribosómicas Pequeñas Bacterianas/fisiologíaRESUMEN
The structural basis of the tRNA selection process is investigated by cryo-electron microscopy of ribosomes programmed with UGA codons and incubated with ternary complex (TC) containing the near-cognate Trp-tRNA(Trp) in the presence of kirromycin. Going through more than 350 000 images and employing image classification procedures, we find â¼8% in which the TC is bound to the ribosome. The reconstructed 3D map provides a means to characterize the arrangement of the near-cognate aa-tRNA with respect to elongation factor Tu (EF-Tu) and the ribosome, as well as the domain movements of the ribosome. One of the interesting findings is that near-cognate tRNA's acceptor stem region is flexible and CCA end becomes disordered. The data bring direct structural insights into the induced-fit mechanism of decoding by the ribosome, as the analysis of the interactions between small and large ribosomal subunit, aa-tRNA and EF-Tu and comparison with the cognate case (UGG codon) offers clues on how the conformational signals conveyed to the GTPase differ in the two cases.
Asunto(s)
Codón , Factor Tu de Elongación Peptídica/química , Biosíntesis de Proteínas , ARN de Transferencia/química , Ribosomas/química , Microscopía por Crioelectrón , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Moleculares , Factor Tu de Elongación Peptídica/metabolismo , ARN de Transferencia/metabolismo , ARN de Transferencia/ultraestructura , Ribosomas/metabolismo , Ribosomas/ultraestructuraRESUMEN
The bacterial stringent response links nutrient starvation with the transcriptional control of genes. This process is initiated by the stringent factor RelA, which senses the presence of deacylated tRNA in the ribosome as a symptom of amino-acid starvation to synthesize the alarmone (p)ppGpp. Here we report a cryo-EM study of RelA bound to ribosomes bearing cognate, deacylated tRNA in the A-site. The data show that RelA on the ribosome stabilizes an unusual distorted form of the tRNA, with the acceptor arm making contact with RelA and far from its normal location in the peptidyl transferase centre.
Asunto(s)
Proteínas de Escherichia coli/química , ARN de Transferencia/química , Ribosomas/metabolismo , Factor de Transcripción ReIA/química , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , ARN de Transferencia/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Factor de Transcripción ReIA/metabolismoRESUMEN
Cryo-EM analysis of a wild-type Escherichia coli pretranslocational sample has revealed the presence of previously unseen intermediate substates of the bacterial ribosome during the first phase of translocation, characterized by intermediate intersubunit rotations, L1 stalk positions, and tRNA configurations. Furthermore, we describe the domain rearrangements in quantitative terms, which has allowed us to characterize the processivity and coordination of the conformational reorganization of the ribosome, along with the associated changes in tRNA ribosome-binding configuration. The results are consistent with the view of the ribosome as a molecular machine employing Brownian motion to reach a functionally productive state via a series of substates with incremental changes in conformation.
Asunto(s)
Biosíntesis de Proteínas , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Sitios de Unión , Microscopía por Crioelectrón , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Sustancias Macromoleculares/química , Sustancias Macromoleculares/ultraestructura , Modelos Moleculares , Conformación Molecular , ARN Mensajero/genética , ARN Mensajero/ultraestructura , ARN de Transferencia/genética , ARN de Transferencia/ultraestructura , Ribosomas/ultraestructura , TermodinámicaRESUMEN
High-resolution structures at different stages, as well as biochemical, single molecule and computational approaches have highlighted the elasticity of tRNA molecules when bound to the ribosome. It is well acknowledged that the inherent structural flexibility of the tRNA lies at the heart of the protein synthesis process. Here, we review the recent advances and describe considerations that the conformational changes of the tRNA molecules offer about the mechanisms grounded in translation.
Asunto(s)
ARN de Transferencia/química , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Biosíntesis de ProteínasRESUMEN
Formation of the 30S initiation complex (30S IC) is an important checkpoint in regulation of gene expression. The selection of mRNA, correct start codon, and the initiator fMet-tRNA(fMet) requires the presence of three initiation factors (IF1, IF2, IF3) of which IF3 and IF1 control the fidelity of the process, while IF2 recruits fMet-tRNA(fMet). Here we present a cryo-EM reconstruction of the complete 30S IC, containing mRNA, fMet-tRNA(fMet), IF1, IF2, and IF3. In the 30S IC, IF2 contacts IF1, the 30S subunit shoulder, and the CCA end of fMet-tRNA(fMet), which occupies a novel P/I position (P/I1). The N-terminal domain of IF3 contacts the tRNA, whereas the C-terminal domain is bound to the platform of the 30S subunit. Binding of initiation factors and fMet-tRNA(fMet) induces a rotation of the head relative to the body of the 30S subunit, which is likely to prevail through 50S subunit joining until GTP hydrolysis and dissociation of IF2 take place. The structure provides insights into the mechanism of mRNA selection during translation initiation.
Asunto(s)
Factores de Iniciación de Péptidos/química , Subunidades Ribosómicas Pequeñas Bacterianas/ultraestructura , Microscopía por Crioelectrón , Escherichia coli/genética , Modelos Moleculares , Conformación Molecular , Factores de Iniciación de Péptidos/ultraestructura , ARN Mensajero/metabolismo , ARN de Transferencia de Metionina/metabolismoRESUMEN
The accuracy of ribosomal translation is achieved by an initial selection and a proofreading step, mediated by EF-Tu, which forms a ternary complex with aminoacyl(aa)-tRNA. To study the binding modes of different aa-tRNAs, we compared cryo-EM maps of the kirromycin-stalled ribosome bound with ternary complexes containing Phe-tRNA(Phe), Trp-tRNA(Trp), or Leu-tRNA(LeuI). The three maps suggest a common binding manner of cognate aa-tRNAs in their specific binding with both the ribosome and EF-Tu. All three aa-tRNAs have the same 'loaded spring' conformation with a kink and twist between the D-stem and anticodon stem. The three complexes are similarly integrated in an interaction network, extending from the anticodon loop through h44 and protein S12 to the EF-Tu-binding CCA end of aa-tRNA, proposed to signal cognate codon-anticodon interaction to the GTPase centre and tune the accuracy of aa-tRNA selection.
Asunto(s)
Factor Tu de Elongación Peptídica/química , Factor Tu de Elongación Peptídica/metabolismo , Estructura Cuaternaria de Proteína , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Ribosomas/química , Ribosomas/metabolismo , Microscopía por Crioelectrón , Modelos Moleculares , Conformación de Ácido Nucleico , Inhibidores de la Síntesis de la Proteína/farmacología , Piridonas/farmacologíaRESUMEN
The ribosome is a complex macromolecular machine that translates the message encoded in the messenger RNA and synthesizes polypeptides by linking the individual amino acids carried by the cognate transfer RNAs (tRNAs). The protein elongation cycle, during which the tRNAs traverse the ribosome in a coordinated manner along a path of more than 100 A, is facilitated by large-scale rearrangements of the ribosome. These rearrangements go hand in hand with conformational changes of tRNA as well as elongation factors EF-Tu and EF-G - GTPases that catalyze tRNA delivery and translocation, respectively. This review focuses on the structural data related to the dynamics of the ribosomal machinery, which are the basis, in conjunction with existing biochemical, kinetic, and fluorescence resonance energy transfer data, of our knowledge of the decoding and translocation steps of protein elongation.
Asunto(s)
Factor G de Elongación Peptídica/metabolismo , Factor Tu de Elongación Peptídica/metabolismo , Biosíntesis de Proteínas/fisiología , ARN de Transferencia/metabolismo , Ribosomas/fisiología , Modelos Biológicos , Factor G de Elongación Peptídica/química , Factor Tu de Elongación Peptídica/química , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN de Transferencia/química , Ribosomas/químicaRESUMEN
Understanding protein synthesis in bacteria and humans is important for understanding the origin of many human diseases and devising treatments for them. Over the past decade, the field of structural biology has made significant advances in the visualisation of the molecular machinery involved in protein synthesis. It is now possible to discern, at least in outline, the way that interlocking ribosomal components and factors adapt their conformations throughout this process. The determination of structures in various functional contexts, along with the application of kinetic and fluorescent resonance energy transfer approaches to the problem, has given researchers the frame of reference for what remains as the greatest challenge: the complete dynamic portrait of protein synthesis in the cell.
Asunto(s)
Biosíntesis de Proteínas/fisiología , Ribosomas/química , Ribosomas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Microscopía Electrónica de Rastreo , Modelos Biológicos , Conformación Proteica , Proteínas/química , Proteínas/metabolismoRESUMEN
The existence of similar folds among major structural subunits of viral capsids has shown unexpected evolutionary relationships suggesting common origins irrespective of the capsids' host life domain. Tailed bacteriophages are emerging as one such family, and we have studied the possible existence of the HK97-like fold in bacteriophage T7. The procapsid structure at approximately 10 A resolution was used to obtain a quasi-atomic model by fitting a homology model of the T7 capsid protein gp10 that was based on the atomic structure of the HK97 capsid protein. A number of fold similarities, such as the fitting of domains A and P into the L-shaped procapsid subunit, are evident between both viral systems. A different feature is related to the presence of the amino-terminal domain of gp10 found at the inner surface of the capsid that might play an important role in the interaction of capsid and scaffolding proteins.
Asunto(s)
Bacteriófago T7/química , Evolución Biológica , Cápside/química , Secuencia de Aminoácidos , Bacteriófago T7/genética , ADN , Datos de Secuencia Molecular , Unión Proteica , Pliegue de Proteína , Estructura Terciaria de ProteínaRESUMEN
Bypassing is a recoding event that leads to the translation of two distal open reading frames into a single polypeptide chain. We present the structure of a translating ribosome stalled at the bypassing take-off site of gene 60 of bacteriophage T4. The nascent peptide in the exit tunnel anchors the P-site peptidyl-tRNAGly to the ribosome and locks an inactive conformation of the peptidyl transferase center (PTC). The mRNA forms a short dynamic hairpin in the decoding site. The ribosomal subunits adopt a rolling conformation in which the rotation of the small subunit around its long axis causes the opening of the A-site region. Together, PTC conformation and mRNA structure safeguard against premature termination and read-through of the stop codon and reconfigure the ribosome to a state poised for take-off and sliding along the noncoding mRNA gap.
Asunto(s)
Biosíntesis de Proteínas , Ribosomas/metabolismo , Modelos Moleculares , Péptidos/química , Péptidos/metabolismo , Conformación Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismo , Ribosomas/química , Relación Estructura-ActividadRESUMEN
Potyviruses constitute the second largest genus of plant viruses and cause important economic losses in a large variety of crops; however, the atomic structure of their particles remains unknown. Infective potyvirus virions are long flexuous filaments where coat protein (CP) subunits assemble in helical mode bound to a monopartite positive-sense single-stranded RNA [(+)ssRNA] genome. We present the cryo-electron microscopy (cryoEM) structure of the potyvirus watermelon mosaic virus at a resolution of 4.0 Å. The atomic model shows a conserved fold for the CPs of flexible filamentous plant viruses, including a universally conserved RNA binding pocket, which is a potential target for antiviral compounds. This conserved fold of the CP is widely distributed in eukaryotic viruses and is also shared by nucleoproteins of enveloped viruses with segmented (-)ssRNA (negative-sense ssRNA) genomes, including influenza viruses.
Asunto(s)
Sitios de Unión , Potyvirus/ultraestructura , Pliegue de Proteína , Proteínas de Unión al ARN/química , Proteínas Virales/química , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Microscopía por Crioelectrón , Modelos Moleculares , Motivos de Nucleótidos , Unión Proteica , Conformación Proteica , ARN Viral/química , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Virales/metabolismoRESUMEN
The three-dimensional structure of the bacteriophage T7 head-to-tail connector has been obtained at 8A resolution using cryo-electron microscopy and single-particle analysis from purified recombinant connectors. The general morphology of the T7 connector is that of a 12-folded toroidal homopolymer with a channel that runs along the longitudinal axis of the particle. The structure of the T7 connector reveals many structural similarities with the connectors from other bacteriophages. Docking of the atomic structure of the varphi29 connector into the three-dimensional reconstruction of T7 connector reveals that the narrow, distal region of the two oligomers are almost identical. This region of the varphi29 connector has been suggested to be involved in DNA translocation, and is composed of an alpha-beta-alpha-beta-beta-alpha motif. A search for alpha-helices in the same region of the T7 three-dimensional map has located three alpha-helices in approximately the same position as those of the varphi29 connector. A comparison of the predicted secondary structure of several bacteriophage connectors, including among others T7, varphi29, P22 and SPP1, reveals that, despite the lack of sequence homology, they seem to contain the same alpha-beta-alpha-beta-beta-alpha motif as that present in the varphi29 connector. These results allow us to suggest a common architecture related to a basic component of the DNA translocating machinery for several viruses.
Asunto(s)
Bacteriófago T7/química , Bacteriófago T7/ultraestructura , ADN Viral/metabolismo , Proteínas Virales/química , Proteínas Virales/ultraestructura , Microscopía por Crioelectrón , Modelos Moleculares , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología Estructural de Proteína , Proteínas Virales/aislamiento & purificación , Proteínas Virales/metabolismoRESUMEN
Flexible filamentous viruses include economically important plant pathogens. Their viral particles contain several hundred copies of a helically arrayed coat protein (CP) protecting a (+)ssRNA. We describe here a structure at 3.9 Å resolution, from electron cryomicroscopy, of Pepino mosaic virus (PepMV), a representative of the genus Potexvirus (family Alphaflexiviridae). Our results allow modeling of the CP and its interactions with viral RNA. The overall fold of PepMV CP resembles that of nucleoproteins (NPs) from the genus Phlebovirus (family Bunyaviridae), a group of enveloped (-)ssRNA viruses. The main difference between potexvirus CP and phlebovirus NP is in their C-terminal extensions, which appear to determine the characteristics of the distinct multimeric assemblies - a flexuous, helical rod or a loose ribonucleoprotein. The homology suggests gene transfer between eukaryotic (+) and (-)ssRNA viruses.
Asunto(s)
Proteínas de la Cápside/ultraestructura , Potexvirus/ultraestructura , Proteínas de la Cápside/metabolismo , Microscopía por Crioelectrón , Modelos Moleculares , Unión Proteica , Pliegue de Proteína , ARN Viral/metabolismoRESUMEN
The double-stranded DNA bacteriophages are good model systems to understand basic biological processes such as the macromolecular interactions that take place during the virus assembly and maturation, or the behavior of molecular motors that function during the DNA packaging process. Using cryoelectron microscopy and single-particle methodology, we have determined the structures of two phage T7 assemblies produced during its morphogenetic process, the DNA-free prohead and the mature virion. The first structure reveals a complex assembly in the interior of the capsid, which involves the scaffolding, and the core complex, which plays an important role in DNA packaging and is located in one of the phage vertices. The reconstruction of the mature virion reveals important changes in the shell, now much larger and thinner, the disappearance of the scaffolding structure, and important rearrangements of the core complex, which now protrudes the shell and interacts with the tail. Some of these changes must originate by the pressure exerted by the DNA in the interior of the head.