Jaime Martín Benito
ULTRASTRUCTURE OF VIRUS AND MOLECULAR AGGREGATES
The main research line of our group is the study of the influenza A ribonucleoproteins (RNPs) that conform the virus nucleocapsid.
Los plásmidos son moléculas de ADN circulares presentes en bacterias que se replican (duplican) de forma independiente del cromosoma bacteriano. La transmisión de plásmidos entre bacterias puede suponer la ganancia o la perdida de una función determinada en la célula receptora. Por ejemplo, algunos genes contenidos en plásmidos son capaces de conferir resistencia a antibióticos, y la transmisión de estos genes puede ser beneficiosa para la supervivencia bacteriana, ya que se convertirán en resistentes a esos antibióticos.
El sistema más habitual de duplicación de estos plásmidos se llama replicación por circulo rodante (RCR). Este proceso requiere la unión de la proteína Rep a una secuencia determinada del ADN, el corte de una de las cadenas del ADN y la separación de la doble cadena a medida que la helicasa PcrA se mueve por una de ellas. Sin embargo, el mecanismo por el cual se inicia la replicación y se desenrolla la doble hélice del ADN no se conoce con detalle.
Investigadores del Centro Nacional de Biotecnología, en colaboración con un grupo de científicos de la Universidad de Pittsburgh (Estados Unidos) han sido capaces de observar a nivel molecular la interacción de las proteínas Rep y PcrA en el proceso de replicación de plásmidos portadores de genes de resistencia a antibióticos.
Nucleic Acids Res. 2020 Jan 13. pii: gkz1200.
Carrasco C, Pastrana CL, Aicart-Ramos C, Leuba SH, Khan SA, Moreno-Herrero F.
Abstract
The rolling-circle replication is the most common mechanism for the replication of small plasmids carrying antibiotic resistance genes in Gram-positive bacteria. It is initiated by the binding and nicking of double-stranded origin of replication by a replication initiator protein (Rep). Duplex unwinding is then performed by the PcrA helicase, whose processivity is critically promoted by its interaction with Rep. How Rep and PcrA proteins interact to nick and unwind the duplex is not fully understood. Here, we have used magnetic tweezers to monitor PcrA helicase unwinding and its relationship with the nicking activity of Staphylococcus aureus plasmid pT181 initiator RepC. Our results indicate that PcrA is a highly processive helicase prone to stochastic pausing, resulting in average translocation rates of 30 bp s-1, while a typical velocity of 50 bp s-1 is found in the absence of pausing. Single-strand DNA binding protein did not affect PcrA translocation velocity but slightly increased its processivity. Analysis of the degree of DNA supercoiling required for RepC nicking, and the time between RepC nicking and DNA unwinding, suggests that RepC and PcrA form a protein complex on the DNA binding site before nicking. A comprehensive model that rationalizes these findings is presented.
doi: 10.1093/nar/gkz1200.
Nat Commun. 2020 Jan 2;11(1):55.
Vilas JL, Tagare HD, Vargas J, Carazo JM, Sorzano COS.
Abstract
The introduction of local resolution has enormously helped the understanding of cryo-EM maps. Still, for any given pixel it is a global, aggregated value, that makes impossible the individual analysis of the contribution of the different projection directions. We introduce MonoDir, a fully automatic, parameter-free method that, starting only from the final cryo-EM map, decomposes local resolution into the different projection directions, providing a detailed level of analysis of the final map. Many applications of directional local resolution are possible, and we concentrate here on map quality and validation.
doi: 10.1038/s41467-019-13742-w.
Muchas macromoléculas biológicas utilizan sistemas de control alostérico para ensamblarse, desensamblarse o cambiar de conformación. Así, cambios en una región de la molécula afectan al comportamiento y la función de la misma en otras regiones. Por ejemplo, la unión de un ligando produce un cambio conformacional que activa o inactiva una enzima.
El desarrollo de moléculas sintéticas que tengan este tipo de propiedades resulta muy complejo y hasta ahora los esfuerzos invertidos en este área solo han conseguido unos pocos resultados exitosos. De hecho, en el caso concreto de la creación de nuevos ensamblajes protéicos de diseño, los resultados hasta ahora se han basado en un sistema tipo puzzle, donde la superficie de cada una de las piezas rígidas que se van a ensamblar es modificada para producir una interacción estable. Ahora, investigadores del Centro Nacional de Biotecnología, en colaboración con el IMDEA Nanociencia, el Centro de Investigaciones Biológicas y las Universidades de Granada y California han desarrollado un modelo que permite controlar de forma alostérica la formación y ensamblaje de proteínas mediante la inclusión de interruptores moleculares formados gracias a cambios en la estructura tridimensional de la proteína.
Sachse M, Fernández de Castro I, Tenorio R, Risco C.
Abstract
Transmission electron microscopy (TEM) has been crucial to study viral infections. As a result of recent advances in light and electron microscopy, we are starting to be aware of the variety of structures that viruses assemble inside cells. Viruses often remodel cellular compartments to build their replication factories. Remarkably, viruses are also able to induce new membranes and new organelles. Here we revise the most relevant imaging technologies to study the biogenesis of viral replication organelles. Live cell microscopy, correlative light and electron microscopy, cryo-TEM, and three-dimensional imaging methods are unveiling how viruses manipulate cell organization. In particular, methods for molecular mapping in situ in two and three dimensions are revealing how macromolecular complexes build functional replication complexes inside infected cells. The combination of all these imaging approaches is uncovering the viral life cycle events with a detail never seen before.
doi: 10.1016/bs.aivir.2019.07.005.
Nucleic Acids Res. 2019 Aug 9.
Martín-González N, Hernando-Pérez M, Condezo GN, Pérez-Illana M, Šiber A, Reguera D, Ostapchuk P, Hearing P6, San Martín C, de Pablo PJ.
Abstract
Some viruses package dsDNA together with large amounts of positively charged proteins, thought to help condense the genome inside the capsid with no evidence. Further, this role is not clear because these viruses have typically lower packing fractions than viruses encapsidating naked dsDNA. In addition, it has recently been shown that the major adenovirus condensing protein (polypeptide VII) is dispensable for genome encapsidation. Here, we study the morphology and mechanics of adenovirus particles with (Ad5-wt) and without (Ad5-VII-) protein VII. Ad5-VII- particles are stiffer than Ad5-wt, but DNA-counterions revert this difference, indicating that VII screens repulsive DNA-DNA interactions. Consequently, its absence results in increased internal pressure. The core is slightly more ordered in the absence of VII and diffuses faster out of Ad5-VII- than Ad5-wt fractured particles. In Ad5-wt unpacked cores, dsDNA associates in bundles interspersed with VII-DNA clusters. These results indicate that protein VII condenses the adenovirus genome by combining direct clustering and promotion of bridging by other core proteins. This condensation modulates the virion internal pressure and DNA release from disrupted particles, which could be crucial to keep the genome protected inside the semi-disrupted capsid while traveling to the nuclear pore.
doi: 10.1093/nar/gkz687
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