RESUMEN
Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Proteínas , Transferencia Resonante de Energía de Fluorescencia/métodos , Reproducibilidad de los Resultados , Proteínas/química , Conformación Molecular , LaboratoriosRESUMEN
RecA-mediated homologous recombination (HR) is a key mechanism for genome maintenance and plasticity in bacteria. It proceeds through RecA assembly into a dynamic filament on ssDNA, the presynaptic filament, which mediates DNA homology search and ordered DNA strand exchange. Here, we combined structural, single molecule and biochemical approaches to characterize the ATP-dependent assembly mechanism of the presynaptic filament of RecA from Streptococcus pneumoniae (SpRecA), in comparison to the Escherichia coli RecA (EcRecA) paradigm. EcRecA polymerization on ssDNA is assisted by the Single-Stranded DNA Binding (SSB) protein, which unwinds ssDNA secondary structures that block EcRecA nucleofilament growth. We report by direct microscopic analysis of SpRecA filamentation on ssDNA that neither of the two paralogous pneumococcal SSBs could assist the extension of SpRecA nucleopolymers. Instead, we found that the conserved RadA helicase promotes SpRecA nucleofilamentation in an ATP-dependent manner. This allowed us to solve the atomic structure of such a long native SpRecA nucleopolymer by cryoEM stabilized with ATPγS. It was found to be equivalent to the crystal structure of the EcRecA filament with a marked difference in how RecA mediates nucleotide orientation in the stretched ssDNA. Then, our results show that SpRecA and EcRecA HR activities are different, in correlation with their distinct ATP-dependent ssDNA binding modes.
Asunto(s)
Rec A Recombinasas , Streptococcus pneumoniae , Adenosina Trifosfato/metabolismo , ADN/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Rec A Recombinasas/metabolismo , Rec A Recombinasas/ultraestructura , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Microscopía por CrioelectrónRESUMEN
Transcription termination mediated by the ring-shaped, ATP-dependent Rho motor is a multipurpose regulatory mechanism specific to bacteria and constitutes an interesting target for the development of new antibiotics. Although Rho-dependent termination can punctuate gene expression or contribute to the protection of the genome at hundreds of sites within a given bacterium, its exact perimeter and site- or species-specific features remain insufficiently characterized. New advanced approaches are required to explore thoroughly the diversity of Rho-dependent terminators and the complexity of associated mechanisms. Current in vitro analyses of Rho-dependent termination rely on radiolabeling, gel electrophoresis, and phosphorimaging of transcription reaction products and are thus hazardous, inconvenient, and low-throughput. To address these limitations, we have developed the first in vitro assay using a fluorescence detection modality to study Rho-dependent transcription termination. This powerful experimental tool accurately estimates terminator strengths in a matter of minutes and is optimized for a microplate reader format allowing multiplexed characterization of putative terminator sequences and mechanisms or high-throughput screening of new drugs targeting Rho-dependent termination.
Asunto(s)
Bioquímica/métodos , Colorantes Fluorescentes , Factor Rho/genética , Terminación de la Transcripción Genética , Sondas Moleculares/genética , Factor Rho/metabolismo , Espectrometría de Fluorescencia , p-Dimetilaminoazobenceno/análogos & derivadosRESUMEN
Conjugation of fluorescent dyes to proteins-a prerequisite for the study of conformational dynamics by single-molecule (sm) FRET-can lead to substantial changes in a dye's photophysical properties, ultimately biasing the determination of inter-dye distances. In particular, cyanine dyes and their derivatives, the most commonly used dyes in smFRET experiments, exhibit such behavior. To overcome this, we developed a general strategy to equip proteins site-specifically with FRET pairs through chemoselective reactions with two distinct noncanonical amino acids simultaneously incorporated through genetic code expansion in Escherichia coli. Application of this technique to human NADPH-cytochrome P450 reductase (CPR) demonstrated the importance of homogenously labeled samples for accurate determination of FRET efficiencies and unveiled the effect of NADP+ on the ionic-strength-dependent modulation of the conformational equilibrium of CPR. Thanks to its generality and accuracy, the presented methodology establishes a new benchmark for deciphering of complex molecular dynamics in single molecules.
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Transferencia Resonante de Energía de Fluorescencia/métodos , NADPH-Ferrihemoproteína Reductasa/química , Imagen Individual de Molécula/métodos , Carbocianinas/química , Clonación Molecular , Escherichia coli/genética , Colorantes Fluorescentes/química , Humanos , Lisina/análogos & derivados , Lisina/química , Microscopía Confocal/métodos , Conformación Molecular , Fenilalanina/análogos & derivados , Fenilalanina/químicaRESUMEN
Antitermination is a regulatory process based on the competitive folding of terminator-antiterminator structures that can form in the leader region of nascent transcripts. In the case of the Bacillus subtilis licS gene involved in ß-glucosides utilization, the binding of the antitermination protein LicT to a short RNA hairpin (RAT) prevents the formation of an overlapping terminator and thereby allows transcription to proceed. Here, we monitored in vitro the competition between termination and antitermination by combining bulk and single-molecule fluorescence-based assays using labeled RNA oligonucleotide constructs of increasing length that mimic the progressive transcription of the terminator invading the antiterminator hairpin. Although high affinity binding is abolished as soon as the antiterminator basal stem is disrupted by the invading terminator, LicT can still bind and promote closing of the partially unfolded RAT hairpin. However, binding no longer occurs once the antiterminator structure has been disrupted by the full-length terminator. Based on these findings, we propose a kinetic competition model for the sequential events taking place at the termination-antitermination site, where LicT needs to capture its RAT target before completion of the terminator to remain tightly bound during RNAP pausing, before finally dissociating irreversibly from the elongated licS transcript.
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Regiones no Traducidas 5' , Proteínas Bacterianas/metabolismo , Pliegue del ARN , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismo , Terminación de la Transcripción Genética , Transferencia Resonante de Energía de Fluorescencia , Conformación de Ácido Nucleico , Transcripción GenéticaRESUMEN
Dimerization is a unique and vital characteristic of retroviral genomes. It is commonly accepted that genomic RNA (gRNA) must be dimeric at the plasma membrane of the infected cells to be packaged during virus assembly. However, where, when and how HIV-1 gRNA find each other and dimerize in the cell are long-standing questions that cannot be answered using conventional approaches. Here, we combine two state-of-the-art, multicolor RNA labeling strategies with two single-molecule microscopy technologies to address these questions. We used 3D-super-resolution structured illumination microscopy to analyze and quantify the spatial gRNA association throughout the cell and monitored the dynamics of RNA-RNA complexes in living-cells by cross-correlation fluctuation analysis. These sensitive and complementary approaches, combined with trans-complementation experiments, reveal for the first time the presence of interacting gRNA in the cytosol, a challenging observation due to the low frequency of these events and their dilution among the bulk of other RNAs, and allow the determination of the subcellular orchestration of the HIV-1 dimerization process.
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Dimerización , VIH-1/genética , Imagenología Tridimensional , Microscopía/métodos , Conformación de Ácido Nucleico , ARN Viral/química , Membrana Celular/metabolismo , Supervivencia Celular , Color , Citosol/metabolismo , Genoma Viral , Células HeLa , Humanos , Hibridación Fluorescente in Situ , ARN Viral/genética , Coloración y Etiquetado , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Rho is a ring-shaped, ATP-fueled motor essential for remodeling transcriptional complexes and R-loops in bacteria. Despite years of research on this fundamental model helicase, key aspects of its mechanism of translocation remain largely unknown. Here, we used single-molecule manipulation and fluorescence methods to directly monitor the dynamics of RNA translocation by Rho. We show that the efficiency of Rho activation is strongly dependent on the force applied on the RNA but that, once active, Rho is able to translocate against a large opposing force (at least 7 pN) by a mechanism involving 'tethered tracking'. Importantly, the ability to directly measure dynamics at the single-molecule level allowed us to determine essential motor properties of Rho. Hence, Rho translocates at a rate of â¼56 nt per second under our experimental conditions, which is 2-5 times faster than velocities measured for RNA polymerase under similar conditions. Moreover, the processivity of Rho (â¼62 nt at a 7 pN opposing force) is large enough for Rho to reach termination sites without dissociating from its RNA loading site, potentially increasing the efficiency of transcription termination. Our findings unambiguously establish 'tethered tracking' as the main pathway for Rho translocation, support 'kinetic coupling' between Rho and RNA polymerase during Rho-dependent termination, and suggest that forces applied on the nascent RNA transcript by cellular substructures could have important implications for the regulation of transcription and its coupling to translation in vivo.
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Factor Rho/metabolismo , Terminación de la Transcripción Genética , Cinética , Modelos Moleculares , Transporte de Proteínas , ARN/metabolismo , Factor Rho/químicaRESUMEN
Chromatin insulators are genetic elements implicated in the organization of chromatin and the regulation of transcription. In Drosophila, different insulator types were characterized by their locus-specific composition of insulator proteins and co-factors. Insulators mediate specific long-range DNA contacts required for the three dimensional organization of the interphase nucleus and for transcription regulation, but the mechanisms underlying the formation of these contacts is currently unknown. Here, we investigate the molecular associations between different components of insulator complexes (BEAF32, CP190 and Chromator) by biochemical and biophysical means, and develop a novel single-molecule assay to determine what factors are necessary and essential for the formation of long-range DNA interactions. We show that BEAF32 is able to bind DNA specifically and with high affinity, but not to bridge long-range interactions (LRI). In contrast, we show that CP190 and Chromator are able to mediate LRI between specifically-bound BEAF32 nucleoprotein complexes in vitro. This ability of CP190 and Chromator to establish LRI requires specific contacts between BEAF32 and their C-terminal domains, and dimerization through their N-terminal domains. In particular, the BTB/POZ domains of CP190 form a strict homodimer, and its C-terminal domain interacts with several insulator binding proteins. We propose a general model for insulator function in which BEAF32/dCTCF/Su(HW) provide DNA specificity (first layer proteins) whereas CP190/Chromator are responsible for the physical interactions required for long-range contacts (second layer). This network of organized, multi-layer interactions could explain the different activities of insulators as chromatin barriers, enhancer blockers, and transcriptional regulators, and suggest a general mechanism for how insulators may shape the organization of higher-order chromatin during cell division.
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Cromatina/genética , ADN/genética , Drosophila melanogaster/genética , Elementos Aisladores/genética , Animales , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Proteínas del Ojo/genética , Redes Reguladoras de Genes , Genoma de los Insectos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Nucleares/genéticaRESUMEN
Förster resonance energy transfer (FRET) plays a key role in biochemistry, organic photovoltaics, and lighting sources. FRET is commonly used as a nanoruler for the short (nanometer) distance between donor and acceptor dyes, yet FRET is equally sensitive to the mutual dipole orientation. The orientation dependence complicates the FRET analysis in biological samples and may even lead to the absence of FRET for perpendicularly oriented donor and acceptor dipoles. Here, we exploit the strongly inhomogeneous and localized fields in plasmonic nanoantennas to open new energy transfer routes, overcoming the limitations from the mutual dipole orientation to ultimately enhance the FRET efficiency. We demonstrate that the simultaneous presence of perpendicular near-field components in the nanoantenna sets favorable energy transfer routes that increase the FRET efficiency up to 50% for nearly perpendicular donor and acceptor dipoles. This new facet of plasmonic nanoantennas enables dipole-dipole energy transfer that would otherwise be forbidden in a homogeneous environment. As such, our approach further increases the applicability of single-molecule FRET over diffraction-limited approaches, with the additional benefits of higher sensitivities and higher concentration ranges toward physiological levels.
RESUMEN
BACKGROUND: Single particle tracking (SPT) is nowadays one of the most popular technique to probe spatio-temporal dynamics of proteins diffusing within the plasma membrane. Indeed membrane components of eukaryotic cells are very dynamic molecules and can diffuse according to different motion modes. Trajectories are often reconstructed frame-by-frame and dynamic properties often evaluated using mean square displacement (MSD) analysis. However, to get statistically significant results in tracking experiments, analysis of a large number of trajectories is required and new methods facilitating this analysis are still needed. RESULTS: In this study we developed a new algorithm based on back-propagation neural network (BPNN) and MSD analysis using a sliding window. The neural network was trained and cross validated with short synthetic trajectories. For simulated and experimental data, the algorithm was shown to accurately discriminate between Brownian, confined and directed diffusion modes within one trajectory, the 3 main of diffusion encountered for proteins diffusing within biological membranes. It does not require a minimum number of observed particle displacements within the trajectory to infer the presence of multiple motion states. The size of the sliding window was small enough to measure local behavior and to detect switches between different diffusion modes for segments as short as 20 frames. It also provides quantitative information from each segment of these trajectories. Besides its ability to detect switches between 3 modes of diffusion, this algorithm is able to analyze simultaneously hundreds of trajectories with a short computational time. CONCLUSION: This new algorithm, implemented in powerful and handy software, provides a new conceptual and versatile tool, to accurately analyze the dynamic behavior of membrane components.
Asunto(s)
Membrana Celular/química , Redes Neurales de la Computación , Algoritmos , Membrana Celular/metabolismo , Difusión , Modelos Biológicos , Movimiento (Física)RESUMEN
ATP-fuelled molecular motors are responsible for rapid and specific transfer of double-stranded DNA during several fundamental processes, such as cell division, sporulation, bacterial conjugation, and viral DNA transport. A dramatic example of intercompartmental DNA transfer occurs during sporulation in Bacillus subtilis, in which two-thirds of a chromosome is transported across a division septum by the SpoIIIE ATPase. Here, we use photo-activated localization microscopy, structured illumination microscopy, and fluorescence fluctuation microscopy to investigate the mechanism of recruitment and assembly of the SpoIIIE pump and the molecular architecture of the DNA translocation complex. We find that SpoIIIE assembles into â¼45 nm complexes that are recruited to nascent sites of septation, and are subsequently escorted by the constriction machinery to the center of sporulation and division septa. SpoIIIE complexes contain 47±20 SpoIIIE molecules, a majority of which are assembled into hexamers. Finally, we show that directional DNA translocation leads to the establishment of a compartment-specific, asymmetric complex that exports DNA. Our data are inconsistent with the notion that SpoIIIE forms paired DNA conducting channels across fused membranes. Rather, our results support a model in which DNA translocation occurs through an aqueous DNA-conducting pore that could be structurally maintained by the divisional machinery, with SpoIIIE acting as a checkpoint preventing membrane fusion until completion of chromosome segregation. Our findings and proposed mechanism, and our unique combination of innovating methodologies, are relevant to the understanding of bacterial cell division, and may illuminate the mechanisms of other complex machineries involved in DNA conjugation and protein transport across membranes.
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Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Conjugación Genética , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Microscopía Fluorescente , Esporas Bacterianas/metabolismoRESUMEN
Rho is a ring-shaped, ATP-dependent RNA helicase/translocase that dissociates transcriptional complexes in bacteria. How RNA recognition is coupled to ATP hydrolysis and translocation in Rho is unclear. Here, we develop and use a new combinatorial approach, called time-resolved Nucleotide Analog Interference Probing (trNAIP), to unmask RNA molecular determinants of catalytic Rho function. We identify a regulatory step in the translocation cycle involving recruitment of the 2'-hydroxyl group of the incoming 3'-RNA nucleotide by a Rho subunit. We propose that this step arises from the intrinsic weakness of one of the subunit interfaces caused by asymmetric, split-ring arrangement of primary RNA tethers around the Rho hexamer. Translocation is at highest stake every seventh nucleotide when the weak interface engages the incoming 3'-RNA nucleotide or breaks, depending on RNA threading constraints in the Rho pore. This substrate-governed, 'test to run' iterative mechanism offers a new perspective on how a ring-translocase may function or be regulated. It also illustrates the interest and versatility of the new trNAIP methodology to unveil the molecular mechanisms of complex RNA-based systems.
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Proteínas Bacterianas/metabolismo , Factor Rho/metabolismo , Proteínas Bacterianas/química , ADN/química , ADN/metabolismo , Técnicas de Sonda Molecular , ARN/química , ARN/metabolismo , ARN Helicasas/química , ARN Helicasas/metabolismo , Factor Rho/químicaRESUMEN
SpoIIIE/FtsK are membrane-anchored, ATP-fuelled, directional motors responsible for chromosomal segregation in bacteria. Directionality in these motors is governed by interactions between specialized sequence-recognition modules (SpoIIIE-γ/FtsK-γ) and highly skewed chromosomal sequences (SRS/KOPS). Using a new combination of ensemble and single-molecule methods, we dissect the series of steps required for SRS localization and motor activation. First, we demonstrate that SpoIIIE/DNA association kinetics are sequence independent, with binding specificity being uniquely determined by dissociation. Next, we show by single-molecule and modelling methods that hexameric SpoIIIE binds DNA non-specifically and finds SRS by an ATP-independent target search mechanism, with ensuing oligomerization and binding of SpoIIIE-γ to SRS triggering motor stimulation. Finally, we propose a new model that provides an entirely new interpretation of previous observations for the origin of SRS/KOPS-directed translocation by SpoIIIE/FtsK.
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Proteínas Bacterianas/química , ADN Bacteriano/genética , Anisotropía , Proteínas Bacterianas/fisiología , Secuencia de Bases , Sitios de Unión , ADN Bacteriano/química , Cinética , Microscopía de Fuerza Atómica , Modelos Moleculares , Unión Proteica , Transporte de Proteínas , Espectrometría de FluorescenciaRESUMEN
The control of transcription termination by RNA-binding proteins that modulate RNA-structures is an important regulatory mechanism in bacteria. LicT and SacY from Bacillus subtilis prevent the premature arrest of transcription by binding to an anti-terminator RNA hairpin that overlaps an intrinsic terminator located in the 5'-mRNA leader region of the gene to be regulated. In order to investigate the molecular determinants of this anti-termination/termination balance, we have developed a fluorescence-based nucleic acids system that mimics the competition between the LicT or SacY anti-terminator targets and the overlapping terminators. Using Förster Resonance Energy Transfer on single diffusing RNA hairpins, we could monitor directly their opening or closing state, and thus investigate the effects on this equilibrium of the binding of anti-termination proteins or terminator-mimicking oligonucleotides. We show that the anti-terminator hairpins adopt spontaneously a closed structure and that their structural dynamics is mainly governed by the length of their basal stem. The induced stability of the anti-terminator hairpins determines both the affinity and specificity of the anti-termination protein binding. Finally, we show that stabilization of the anti-terminator hairpin, by an extended basal stem or anti-termination protein binding can efficiently counteract the competing effect of the terminator-mimic.
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Proteínas Bacterianas/metabolismo , ARN Bacteriano/química , Proteínas de Unión al ARN/metabolismo , Regiones Terminadoras Genéticas , Factores de Transcripción/metabolismo , Bacillus subtilis/genética , Transferencia Resonante de Energía de Fluorescencia , Conformación de Ácido Nucleico , Unión Proteica , Pliegue del ARN , ARN Bacteriano/metabolismo , Electricidad Estática , Terminación de la Transcripción GenéticaRESUMEN
Pulsed Interleaved Excitation (PIE) improves fluorescence cross-correlation spectroscopy (FCCS) and single pair Förster Resonance Energy Transfer (spFRET) measurements, by correlating each detected photon to the excitation source that generated it. It relies on the interleaving of two picosecond laser sources and time correlated single photon counting (TCSPC) detection. Here, we present an optical configuration based on a commercial supercontinuum laser, which generates multicoulour interleaved picosecond pulses with arbitrary spacing and wavelengths within the visible spectrum. This simple, yet robust configuration can be used as a versatile source for PIE experiments, as an alternative to an array of picosecond lasers and drivers.
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Transferencia Resonante de Energía de Fluorescencia/instrumentación , Iluminación/instrumentación , Diseño de Equipo , Análisis de Falla de EquipoRESUMEN
Allosteric modulators bear great potential to fine-tune neurotransmitter action. Promising targets are metabotropic glutamate (mGlu) receptors, which are associated with numerous brain diseases. Orthosteric and allosteric ligands act in synergy to control the activity of these multidomain dimeric GPCRs. Here, we analyzed the effect of such molecules on the concerted conformational changes of full-length mGlu2 at the single-molecule level. We first established FRET sensors through genetic code expansion combined with click chemistry to monitor conformational changes on live cells. We then used single-molecule FRET and show that orthosteric agonist binding leads to the stabilization of most of the glutamate binding domains in their closed state, while the reorientation of the dimer into the active state remains partial. Allosteric modulators, interacting with the transmembrane domain, are required to stabilize the fully reoriented active dimer. These results illustrate how concerted conformational changes within multidomain proteins control their activity, and how these are modulated by allosteric ligands.
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Receptores de Glutamato Metabotrópico , Regulación Alostérica , Ligandos , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/metabolismo , GlutamatosRESUMEN
Partitioning of membrane proteins into various types of microdomains is crucial for many cellular functions. Tetraspanin-enriched microdomains (TEMs) are a unique type of protein-based microdomain, clearly distinct from membrane rafts, and important for several cellular processes such as fusion, migration and signaling. Paradoxically, HIV-1 assembly/egress occurs at TEMs, yet the viral particles also incorporate raft lipids. Using different quantitative microscopy approaches, we investigated the dynamic relationship between TEMs, membrane rafts and HIV-1 exit sites, focusing mainly on the tetraspanin CD9. Our results show that clustering of CD9 correlates with multimerization of the major viral structural component, Gag, at the plasma membrane. CD9 exhibited confined behavior and reduced lateral mobility at viral assembly sites, suggesting that Gag locally traps tetraspanins. In contrast, the raft lipid GM1 and the raft-associated protein CD55, while also recruited to assembly/budding sites, were only transiently trapped in these membrane areas. CD9 recruitment and confinement were found to be partially dependent on cholesterol, while those of CD55 were completely dependent on cholesterol. Importantly, our findings support the emerging concept that cellular and viral components, instead of clustering at preexisting microdomain platforms, direct the formation of distinct domains for the execution of specific functions.
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VIH-1/fisiología , Glicoproteínas de Membrana/metabolismo , Microdominios de Membrana/metabolismo , Ensamble de Virus/fisiología , Animales , Chlorocebus aethiops , Recuperación de Fluorescencia tras Fotoblanqueo , Células HeLa , Humanos , Células VeroRESUMEN
Real-time visualization and quantification of viruses released by a cell are crucial to further decipher infection processes. Kinetics studies at the single-cell level will circumvent the limitations of bulk assays with asynchronous virus replication. We have implemented a "viro-fluidic" method, which combines microfluidics and virology at single-cell and single-virus resolutions. As an experimental model, we used standard cell lines producing fluorescent HIV-like particles (VLPs). First, to scale the strategy to the single-cell level, we validated a sensitive flow virometry system to detect VLPs in low concentration samples (≥104 VLPs/mL). Then, this system was coupled to a single-cell trapping device to monitor in real-time the VLPs released, one at a time, from single cells under cell culture conditions. Our results revealed an average production rate of 50 VLPs/h/cell similar to the rate estimated for the same cells grown in population. Thus, the virus-producing capacities of the trapped cells were preserved and its real-time monitoring was accurate. Moreover, single-cell analysis revealed a release of VLPs with stochastic bursts with typical time intervals of few minutes, revealing the existence of limiting step(s) in the virus biogenesis process. Our tools can be applied to other pathogens or to extracellular vesicles to elucidate the dissemination mechanisms of these biological nanoparticles.
RESUMEN
The bacterial Rho factor is a ring-shaped motor triggering genome-wide transcription termination and R-loop dissociation. Rho is essential in many species, including in Mycobacterium tuberculosis where rho gene inactivation leads to rapid death. Yet, the M. tuberculosis Rho [MtbRho] factor displays poor NTPase and helicase activities, and resistance to the natural Rho inhibitor bicyclomycin [BCM] that remain unexplained. To address these issues, we solved the cryo-EM structure of MtbRho at 3.3 Šresolution. The MtbRho hexamer is poised into a pre-catalytic, open-ring state wherein specific contacts stabilize ATP in intersubunit ATPase pockets, thereby explaining the cofactor preference of MtbRho. We reveal a leucine-to-methionine substitution that creates a steric bulk in BCM binding cavities near the positions of ATP γ-phosphates, and confers resistance to BCM at the expense of motor efficiency. Our work contributes to explain the unusual features of MtbRho and provides a framework for future antibiotic development.