RESUMEN
Polyadenylate [poly(A)] tail addition to the 3' end of a wide range of RNAs is a highly conserved modification that plays a central role in cellular RNA function. Elements for nuclear expression (ENEs) are cis-acting RNA elements that stabilize poly(A) tails by sequestering them in RNA triplex structures. A crystal structure of a double ENE from a rice hAT transposon messenger RNA complexed with poly(A)28 at a resolution of 2.89 angstroms reveals multiple modes of interaction with poly(A), including major-groove triple helices, extended minor-groove interactions with RNA double helices, a quintuple-base motif that transitions poly(A) from minor-groove associations to major-groove triple helices, and a poly(A) 3'-end binding pocket. Our findings both expand the repertoire of motifs involved in long-range RNA interactions and provide insights into how polyadenylation can protect an RNA's extreme 3' end.
Asunto(s)
Poli A/química , Poliadenilación , Estabilidad del ARN , ARN Mensajero/química , Cristalización , Conformación de Ácido Nucleico , OryzaRESUMEN
In bacteria, the dissociable σ subunit of the RNA polymerase (RNAP) is responsible for initiating RNA synthesis from specific DNA sites. As nascent RNA grows, downstream DNA unwinds and is pulled into the RNAP, causing stress accumulation and initiation complex destabilization. Processive transcription elongation requires at least partial separation of the σ factor from the RNAP core enzyme. Here, we present a series of transcription complexes captured between the early initiation and elongation phases via in-crystal RNA synthesis and cleavage. Crystal structures of these complexes indicate that stress accumulation during transcription initiation is not due to clashing of the growing nascent RNA with the σ3.2 loop, but results from scrunching of the template strand DNA that is contained inside the RNAP by the σ3 domain. Our results shed light on how scrunching of template-strand DNA drives both abortive initiation and σ-RNAP core separation to transition transcription from initiation to elongation.
RESUMEN
The density regulated protein (DENR) forms a stable heterodimer with malignant T-cell-amplified sequence 1 (MCT-1). DENR-MCT-1 heterodimer then participates in regulation of non-canonical translation initiation and ribosomal recycling. The N-terminal domain of DENR interacts with MCT-1 and carries a classical tetrahedral zinc ion-binding site, which is crucial for the dimerization. DENR-MCT-1 binds the small (40S) ribosomal subunit through interactions between MCT-1 and helix h24 of the 18S rRNA, and through interactions between the C-terminal domain of DENR and helix h44 of the 18S rRNA. This later interaction occurs in the vicinity of the P site that is also the binding site for canonical translation initiation factor eIF1, which plays the key role in initiation codon selection and scanning. Sequence homology modeling and a low-resolution crystal structure of the DENR-MCT-1 complex with the human 40S subunit suggests that the C-terminal domain of DENR and eIF1 adopt a similar fold. Here we present the crystal structure of the C-terminal domain of DENR determined at 1.74 Å resolution, which confirms its resemblance to eIF1 and advances our understanding of the mechanism by which DENR-MCT-1 regulates non-canonical translation initiation and ribosomal recycling.
RESUMEN
Pistol ribozymes comprise a class of small, self-cleaving RNAs discovered via comparative genomic analysis. Prior work in the field has probed the kinetics of the cleavage reaction, as well as the influence of various metal ion cofactors that accelerate the process. In the current study, we performed unbiased and unconstrained molecular dynamics simulations from two current high-resolution pistol crystal structures, and we analyzed trajectory data within the context of the currently accepted ribozyme mechanistic framework. Root-mean-squared deviations, radial distribution functions, and distributions of nucleophilic angle-of-attack reveal insights into the potential roles of three magnesium ions with respect to catalysis and overall conformational stability of the molecule. A series of simulation trajectories containing in silico mutations reveal the relatively flexible and partially interchangeable roles of two particular magnesium ions within solvated hydrogen-bonding distances from the catalytic center.
Asunto(s)
Magnesio/química , Simulación de Dinámica Molecular , ARN Catalítico/química , Biocatálisis , Iones/química , Iones/metabolismo , Magnesio/metabolismo , ARN Catalítico/metabolismoRESUMEN
To address drug resistance to HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs), a series of novel diarylpyrimidine (DAPY) derivatives targeting "tolerant region I" and "tolerant region II" of the NNRTIs binding pocket (NNIBP) were designed utilizing a structure-guided scaffold-hopping strategy. The dihydrofuro[3,4- d]pyrimidine derivatives 13c2 and 13c4 proved to be exceptionally potent against a wide range of HIV-1 strains carrying single NNRTI-resistant mutations (EC50 = 0.9-8.4 nM), which were remarkably superior to that of etravirine (ETV). Meanwhile, both compounds exhibited comparable activities with ETV toward the virus with double mutations F227L+V106A and K103N+Y181C. Furthermore, the most active compound 13c2 showed favorable pharmacokinetic properties with an oral bioavailability of 30.96% and a half-life of 11.1 h, which suggested that 13c2 is worth further investigation as a novel NNRTI to circumvent drug resistance.
Asunto(s)
Fármacos Anti-VIH/farmacología , Furanos/farmacología , VIH-1/enzimología , Pirimidinas/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Animales , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacocinética , Sitios de Unión , Línea Celular Tumoral , Furanos/síntesis química , Furanos/farmacocinética , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/metabolismo , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Simulación de Dinámica Molecular , Estructura Molecular , Pirimidinas/síntesis química , Pirimidinas/farmacocinética , Ratas Wistar , Inhibidores de la Transcriptasa Inversa/síntesis química , Inhibidores de la Transcriptasa Inversa/farmacocinética , Relación Estructura-ActividadRESUMEN
The density-regulated protein (DENR) and the malignant T cell-amplified sequence 1 (MCT-1/MCTS1) oncoprotein support noncanonical translation initiation, promote translation reinitiation on a specific set of mRNAs with short upstream reading frames, and regulate ribosome recycling. DENR and MCT-1 form a heterodimer, which binds to the ribosome. We determined the crystal structure of the heterodimer formed by human MCT-1 and the N-terminal domain of DENR at 2.0-Å resolution. The structure of the heterodimer reveals atomic details of the mechanism of DENR and MCT-1 interaction. Four conserved cysteine residues of DENR (C34, C37, C44, C53) form a classical tetrahedral zinc ion-binding site, which preserves the structure of the DENR's MCT-1-binding interface that is essential for the dimerization. Substitution of all four cysteines by alanine abolished a heterodimer formation. Our findings elucidate further the mechanism of regulation of DENR-MCT-1 activities in unconventional translation initiation, reinitiation, and recycling.
Asunto(s)
Proteínas de Ciclo Celular/química , Factores Eucarióticos de Iniciación/química , Proteínas Oncogénicas/química , Multimerización de Proteína , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Factores Eucarióticos de Iniciación/genética , Factores Eucarióticos de Iniciación/metabolismo , Humanos , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Estructura Cuaternaria de ProteínaRESUMEN
Rapid generation of drug-resistant mutations in HIV-1 reverse transcriptase (RT), a prime target for anti-HIV therapy, poses a major impediment to effective anti-HIV treatment. Our previous efforts have led to the development of two novel non-nucleoside reverse transcriptase inhibitors (NNRTIs) with piperidine-substituted thiophene[3,2-d]pyrimidine scaffolds, compounds K-5a2 and 25a, which demonstrate highly potent anti-HIV-1 activities and improved resistance profiles compared with etravirine and rilpivirine, respectively. Here, we have determined the crystal structures of HIV-1 wild-type (WT) RT and seven RT variants bearing prevalent drug-resistant mutations in complex with K-5a2 or 25a at ~2 Å resolution. These high-resolution structures illustrate the molecular details of the extensive hydrophobic interactions and the network of main chain hydrogen bonds formed between the NNRTIs and the RT inhibitor-binding pocket, and provide valuable insights into the favorable structural features that can be employed for designing NNRTIs that are broadly active against drug-resistant HIV-1 variants.
Asunto(s)
Infecciones por VIH/tratamiento farmacológico , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/efectos de los fármacos , Pirimidinas/química , Inhibidores de la Transcriptasa Inversa/química , Tiofenos/química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Sitios de Unión , Células Cultivadas , Cristalografía por Rayos X , Diseño de Fármacos , Infecciones por VIH/virología , VIH-1/enzimología , Humanos , Modelos Moleculares , Mutación , Conformación Proteica , Pirimidinas/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Relación Estructura-Actividad , Tiofenos/farmacologíaRESUMEN
Genetic information is translated into proteins by the ribosome. Structural studies of the ribosome and of its complexes with factors and inhibitors have provided invaluable information on the mechanism of protein synthesis. Ribosome inhibitors are among the most successful antimicrobial drugs and constitute more than half of all medicines used to treat infections. However, bacterial infections are becoming increasingly difficult to treat because the microbes have developed resistance to the most effective antibiotics, creating a major public health care threat. This has spurred a renewed interest in structure-function studies of protein synthesis inhibitors, and in few cases, compounds have been developed into potent therapeutic agents against drug-resistant pathogens. In this review, we describe the modes of action of many ribosome-targeting antibiotics, highlight the major resistance mechanisms developed by pathogenic bacteria, and discuss recent advances in structure-assisted design of new molecules.
Asunto(s)
Antibacterianos/farmacología , Ribosomas/efectos de los fármacos , Animales , Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Sitios de Unión , Diseño de Fármacos , Farmacorresistencia Microbiana , Humanos , Modelos Biológicos , Modelos Moleculares , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/química , Inhibidores de la Síntesis de la Proteína/farmacología , Ribosomas/química , Ribosomas/metabolismo , Relación Estructura-ActividadRESUMEN
In bacteria, the activation of gene transcription at many promoters is simple and only involves a single activator. The cyclic adenosine 3',5'-monophosphate receptor protein (CAP), a classic activator, is able to activate transcription independently through two different mechanisms. Understanding the class I mechanism requires an intact transcription activation complex (TAC) structure at a high resolution. Here we report a high-resolution cryo-electron microscopy structure of an intact Escherichia coli class I TAC containing a CAP dimer, a σ70-RNA polymerase (RNAP) holoenzyme, a complete class I CAP-dependent promoter DNA, and a de novo synthesized RNA oligonucleotide. The structure shows how CAP wraps the upstream DNA and how the interactions recruit RNAP. Our study provides a structural basis for understanding how activators activate transcription through the class I recruitment mechanism.
Asunto(s)
Proteína Receptora de AMP Cíclico/química , ARN Polimerasas Dirigidas por ADN/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Factor sigma/química , Activación Transcripcional , Microscopía por Crioelectrón , Proteína Receptora de AMP Cíclico/ultraestructura , ADN Bacteriano/química , ADN Bacteriano/ultraestructura , ARN Polimerasas Dirigidas por ADN/ultraestructura , Proteínas de Escherichia coli/ultraestructura , Regiones Promotoras Genéticas , Factor sigma/ultraestructuraRESUMEN
Protein synthesis is a key process in all living organisms. In eukaryotes, initiation factor 2 (eIF2) plays an important role in translation initiation as it selects and delivers the initiator tRNA to the small ribosomal subunit. Under stress conditions, phosphorylation of the α-subunit of eIF2 downregulates cellular protein synthesis. However, translation of certain mRNAs continues via the eIF2D-dependent non-canonical initiation pathway. The molecular mechanism of this process remains elusive. In addition, eIF2D plays a role in translation re-initiation and ribosome recycling. Currently, there has been no structural information of eIF2D. We have now determined the crystal structure of the C-terminal domains of eIF2D at 1.4-Å resolution. One domain has the fold similar to that of eIF1, which is crucial for the scanning and initiation codon selection. The second domain has a known SWIB/MDM2 fold, which was not observed before in other translation initiation factors. Our structure reveals atomic details of inter-domain interactions in the C-terminal part of eIF2D and sheds light on the possible role of these domains in eIF2D during translation.
Asunto(s)
Factor 2 Eucariótico de Iniciación/química , Iniciación de la Cadena Peptídica Traduccional , Cristalografía por Rayos X , Factor 2 Eucariótico de Iniciación/metabolismo , Humanos , Modelos Moleculares , Conformación Proteica , Dominios ProteicosRESUMEN
The repertoire of the density-regulated protein (DENR) and the malignant T cell-amplified sequence 1 (MCT-1/MCTS1) oncoprotein was recently expanded to include translational control of a specific set of cancer-related mRNAs. DENR and MCT-1 form the heterodimer, which binds to the ribosome and operates at both translation initiation and reinitiation steps, though by a mechanism that is yet unclear. Here, we determined the crystal structure of the human small ribosomal subunit in complex with DENR-MCT-1. The structure reveals the location of the DENR-MCT-1 dimer bound to the small ribosomal subunit. The binding site of the C-terminal domain of DENR on the ribosome has a striking similarity with those of canonical initiation factor 1 (eIF1), which controls the fidelity of translation initiation and scanning. Our findings elucidate how the DENR-MCT-1 dimer interacts with the ribosome and have functional implications for the mechanism of unconventional translation initiation and reinitiation.
Asunto(s)
Proteínas de Ciclo Celular/química , Factores Eucarióticos de Iniciación/química , Proteínas Oncogénicas/química , Ribosomas/química , Cristalografía por Rayos X , Humanos , Estructura Cuaternaria de ProteínaRESUMEN
Small self-cleaving ribozymes have been discovered in all evolutionary domains of life. They can catalyze site-specific RNA cleavage, and as a result, they have relevance in gene regulation. Comparative genomic analysis has led to the discovery of a new class of small self-cleaving ribozymes named Pistol. We report the crystal structure of Pistol at 2.97-Å resolution. Our results suggest that the Pistol ribozyme self-cleavage mechanism likely uses a guanine base in the active site pocket to carry out the phosphoester transfer reaction. The guanine G40 is in close proximity to serve as the general base for activating the nucleophile by deprotonating the 2'-hydroxyl to initiate the reaction (phosphoester transfer). Furthermore, G40 can also establish hydrogen bonding interactions with the nonbridging oxygen of the scissile phosphate. The proximity of G32 to the O5' leaving group suggests that G32 may putatively serve as the general acid. The RNA structure of Pistol also contains A-minor interactions, which seem to be important to maintain its tertiary structure and compact fold. Our findings expand the repertoire of ribozyme structures and highlight the conserved evolutionary mechanism used by ribozymes for catalysis.
Asunto(s)
Autoempalme del ARN Ribosómico/química , Catálisis , Dominio Catalítico , Cationes Bivalentes/metabolismo , Cristalización , Cristalografía por Rayos X , Modelos Moleculares , Conformación de Ácido Nucleico , Oligonucleótidos/metabolismo , Mutación Puntual , Autoempalme del ARN Ribosómico/metabolismo , Especificidad por SustratoRESUMEN
During transcription, RNA polymerase moves downstream along the DNA template and maintains a transcription bubble. Several recent structural studies of transcription complexes with a complete transcription bubble provide new insights into how RNAP couples the nucleotide addition reaction to its directional movement.
Asunto(s)
ADN/genética , Transcripción Genética , ADN/química , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Modelos MolecularesRESUMEN
NusG is an essential transcription factor that plays multiple key regulatory roles in transcription elongation, termination and coupling translation and transcription. The core role of NusG is to enhance transcription elongation and RNA polymerase processivity. Here, we present the structure of Escherichia coli RNA polymerase complexed with NusG. The structure shows that the NusG N-terminal domain (NGN) binds at the central cleft of RNA polymerase surrounded by the ß' clamp helices, the ß protrusion, and the ß lobe domains to close the promoter DNA binding channel and constrain the ß' clamp domain, but with an orientation that is different from the one observed in the archaeal ß' clamp-Spt4/5 complex. The structure also allows us to construct a reliable model of the complete NusG-associated transcription elongation complex, suggesting that the NGN domain binds at the upstream fork junction of the transcription elongation complex, similar to σ2 in the transcription initiation complex, to stabilize the junction, and therefore enhances transcription processivity.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Factores de Elongación de Péptidos/química , Factores de Elongación de Péptidos/metabolismo , Elongación de la Transcripción Genética , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Modelos Biológicos , Modelos Moleculares , Conformación Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Relación Estructura-ActividadRESUMEN
In bacteria, multiple σ factors compete to associate with the RNA polymerase (RNAP) core enzyme to form a holoenzyme that is required for promoter recognition. During transcription initiation RNAP remains associated with the upstream promoter DNA via sequence-specific interactions between the σ factor and the promoter DNA while moving downstream for RNA synthesis. As RNA polymerase repetitively adds nucleotides to the 3'-end of the RNA, a pyrophosphate ion is generated after each nucleotide incorporation. It is currently unknown how the release of pyrophosphate affects transcription. Here we report the crystal structures of E coli transcription initiation complexes (TICs) containing the stress-responsive σ(S) factor, a de novo synthesized RNA oligonucleotide, and a complete transcription bubble (σ(S)-TIC) at about 3.9-Å resolution. The structures show the 3D topology of the σ(S) factor and how it recognizes the promoter DNA, including likely specific interactions with the template-strand residues of the -10 element. In addition, σ(S)-TIC structures display a highly stressed pretranslocated initiation complex that traps a pyrophosphate at the active site that remains closed. The position of the pyrophosphate and the unusual phosphodiester linkage between the two terminal RNA residues suggest an unfinished nucleotide-addition reaction that is likely at equilibrium between nucleotide addition and pyrophosphorolysis. Although these σ(S)-TIC crystals are enzymatically active, they are slow in nucleotide addition, as suggested by an NTP soaking experiment. Pyrophosphate release completes the nucleotide addition reaction and is associated with extensive conformational changes around the secondary channel but causes neither active site opening nor transcript translocation.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/metabolismo , Factor sigma/metabolismo , Transcripción Genética , Modelos Moleculares , Factor sigma/químicaRESUMEN
Cisplatin is a widely prescribed anticancer drug, which triggers cell death by covalent binding to a broad range of biological molecules. Among cisplatin targets, cellular RNAs remain the most poorly characterized molecules. Although cisplatin was shown to inactivate essential RNAs, including ribosomal, spliceosomal and telomeric RNAs, cisplatin binding sites in most RNA molecules are unknown, and therefore it remains challenging to study how modifications of RNA by cisplatin contributes to its toxicity. Here we report a 2.6Å-resolution X-ray structure of cisplatin-modified 70S ribosome, which describes cisplatin binding to the ribosome and provides the first nearly atomic model of cisplatin-RNA complex. We observe nine cisplatin molecules bound to the ribosome and reveal consensus structural features of the cisplatin-binding sites. Two of the cisplatin molecules modify conserved functional centers of the ribosome-the mRNA-channel and the GTPase center. In the mRNA-channel, cisplatin intercalates between the ribosome and the messenger RNA, suggesting that the observed inhibition of protein synthesis by cisplatin is caused by impaired mRNA-translocation. Our structure provides an insight into RNA targeting and inhibition by cisplatin, which can help predict cisplatin-binding sites in other cellular RNAs and design studies to elucidate a link between RNA modifications by cisplatin and cisplatin toxicity.
Asunto(s)
Antineoplásicos/química , Cisplatino/química , ARN Ribosómico/química , Ribosomas/química , Adenina/química , Antibacterianos/química , Antineoplásicos/metabolismo , Sitios de Unión , Cisplatino/metabolismo , Cumarinas/química , Cristalografía por Rayos X , Guanina/química , Modelos Moleculares , Inhibidores de la Síntesis del Ácido Nucleico/química , ARN Ribosómico/metabolismo , Ribosomas/metabolismoRESUMEN
During translation, a plethora of protein factors bind to the ribosome and regulate protein synthesis. Many of those factors are guanosine triphosphatases (GTPases), proteins that catalyze the hydrolysis of guanosine 5'-triphosphate (GTP) to promote conformational changes. Despite numerous studies, the function of elongation factor 4 (EF-4/LepA), a highly conserved translational GTPase, has remained elusive. Here, we present the crystal structure at 2.6-Å resolution of the Thermus thermophilus 70S ribosome bound to EF-4 with a nonhydrolyzable GTP analog and A-, P-, and E-site tRNAs. The structure reveals the interactions of EF-4 with the A-site tRNA, including contacts between the C-terminal domain (CTD) of EF-4 and the acceptor helical stem of the tRNA. Remarkably, EF-4 induces a distortion of the A-site tRNA, allowing it to interact simultaneously with EF-4 and the decoding center of the ribosome. The structure provides insights into the tRNA-remodeling function of EF-4 on the ribosome and suggests that the displacement of the CCA-end of the A-site tRNA away from the peptidyl transferase center (PTC) is functionally significant.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestructura , Factores de Iniciación de Péptidos/química , Factores de Iniciación de Péptidos/ultraestructura , ARN Bacteriano/química , ARN Bacteriano/ultraestructura , ARN de Transferencia/química , ARN de Transferencia/ultraestructura , Sitios de Unión , Simulación por Computador , Simulación del Acoplamiento Molecular , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Motivos de Unión al ARN , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/ultraestructura , RibosomasRESUMEN
With bacterial resistance becoming a serious threat to global public health, antimicrobial peptides (AMPs) have become a promising area of focus in antibiotic research. AMPs are derived from a diverse range of species, from prokaryotes to humans, with a mechanism of action that often involves disruption of the bacterial cell membrane. Proline-rich antimicrobial peptides (PrAMPs) are instead actively transported inside the bacterial cell where they bind and inactivate specific targets. Recently, it was reported that some PrAMPs, such as Bac71 -35, oncocins and apidaecins, bind and inactivate the bacterial ribosome. Here we report the crystal structures of Bac71 -35, Pyrrhocoricin, Metalnikowin and two oncocin derivatives, bound to the Thermus thermophilus 70S ribosome. Each of the PrAMPs blocks the peptide exit tunnel of the ribosome by simultaneously occupying three well characterized antibiotic-binding sites and interferes with the initiation step of translation, thereby revealing a common mechanism of action used by these PrAMPs to inactivate protein synthesis. Our study expands the repertoire of PrAMPs and provides a framework for designing new-generation therapeutics.