Remodelling of Rea1 linker domain drives the removal of assembly factors from pre-ribosomal particles.

The ribosome maturation factor Rea1 (or Midasin) catalyses the removal of assembly factors from large ribosomal subunit precursors and promotes their export from the nucleus to the cytosol. Rea1 consists of nearly 5000 amino-acid residues and belongs to the AAA+ protein family. It consists of a ring of six AAA+ domains from which the ≈1700 amino-acid residue linker emerges that is subdivided into stem, middle and top domains.

The structure of the NuA4-Tip60 complex reveals the mechanism and importance of long-range chromatin modification.

Histone acetylation regulates most DNA transactions and is dynamically controlled by highly conserved enzymes. The only essential histone acetyltransferase (HAT) in yeast, Esa1, is part of the 1-MDa NuA4 complex, which plays pivotal roles in both transcription and DNA-damage repair. NuA4 has the unique capacity to acetylate histone targets located several nucleosomes away from its recruitment site.

Differential impact of ubiquitous and muscle dynamin 2 isoforms in muscle physiology and centronuclear myopathy.

Dynamin 2 mechanoenzyme is a key regulator of membrane remodeling and gain-of-function mutations in its gene cause centronuclear myopathies. Here, we investigate the functions of dynamin 2 isoforms and their associated phenotypes and, specifically, the ubiquitous and muscle-specific dynamin 2 isoforms expressed in skeletal muscle. In cell-based assays, we show that a centronuclear myopathy-related mutation in the ubiquitous but not the muscle-specific dynamin 2 isoform causes increased membrane fission.

In vitro characterization of the full-length human dynein-1 cargo adaptor BicD2.

Cargo adaptors are crucial in coupling motor proteins with their respective cargos and regulatory proteins. BicD2 is a prominent example within the cargo adaptor family. BicD2 is able to recruit the microtubule motor dynein to RNA, viral particles, and nuclei.

BIN1 modulation in vivo rescues dynamin-related myopathy.

The mechanoenzyme dynamin 2 (DNM2) is crucial for intracellular organization and trafficking. is mutated in dominant centronuclear myopathy (DNM2-CNM), a muscle disease characterized by defects in organelle positioning in myofibers. It remains unclear how the in vivo functions of DNM2 are regulated in muscle.

Atomic structure of the SAGA complex and it's interaction with TBP.

The transcription of eukaryotic protein genes is controlled by a plethora of proteins which act together in multi-component complexes to facilitate the DNA dependent RNA polymerase II (Pol II) enzyme to bind to the transcription start site and to generate messenger RNA from the gene's coding sequence. The protein that guides the transcription machinery to the exact transcription start site is called TATA-box Binding Protein, or TBP. TBP is part of two large protein complexes involved in Pol II transcription, TFIID and SAGA.

Structure of SAGA and mechanism of TBP deposition on gene promoters.

SAGA (Spt-Ada-Gcn5-acetyltransferase) is a 19-subunit complex that stimulates transcription via two chromatin-modifying enzymatic modules and by delivering the TATA box binding protein (TBP) to nucleate the pre-initiation complex on DNA, a pivotal event in the expression of protein-encoding genes. Here we present the structure of yeast SAGA with bound TBP. The core of the complex is resolved at 3.

Structural Basis of Transcription: RNA Polymerase Backtracking and Its Reactivation.

Regulatory sequences or erroneous incorporations during DNA transcription cause RNA polymerase backtracking and inactivation in all kingdoms of life. Reactivation requires RNA transcript cleavage. Essential transcription factors (GreA and GreB, or TFIIS) accelerate this reaction.

Structural Basis for NusA Stabilized Transcriptional Pausing.

Transcriptional pausing by RNA polymerases (RNAPs) is a key mechanism to regulate gene expression in all kingdoms of life and is a prerequisite for transcription termination. The essential bacterial transcription factor NusA stimulates both pausing and termination of transcription, thus playing a central role. Here, we report single-particle electron cryo-microscopy reconstructions of NusA bound to paused E.

Structure and Dynamics of a 197 bp Nucleosome in Complex with Linker Histone H1.

Linker histones associate with nucleosomes to promote the formation of higher-order chromatin structure, but the underlying molecular details are unclear. We investigated the structure of a 197 bp nucleosome bearing symmetric 25 bp linker DNA arms in complex with vertebrate linker histone H1. We determined electron cryo-microscopy (cryo-EM) and crystal structures of unbound and H1-bound nucleosomes and validated these structures by site-directed protein cross-linking and hydroxyl radical footprinting experiments.

Covalent Tethering and Residues with Bulky Hydrophobic Side Chains Enable Self-Assembly of Distinct Amyloid Structures.

Polymorphism is a common property of amyloid fibers that complicates their detailed structural and functional studies. Here we report experiments illustrating the chemical principles that enable the formation of amyloid polymorphs with distinct stoichiometric composition. Using appropriate covalent tethering we programmed self-assembly of a model peptide corresponding to the [20-41] fragment of human β2-microglobulin into fibers with either trimeric or dimeric amyloid cores.

Structure of the initiation-competent RNA polymerase I and its implication for transcription.

Eukaryotic RNA polymerase I (Pol I) is specialized in rRNA gene transcription synthesizing up to 60% of cellular RNA. High level rRNA production relies on efficient binding of initiation factors to the rRNA gene promoter and recruitment of Pol I complexes containing initiation factor Rrn3. Here, we determine the cryo-EM structure of the Pol I-Rrn3 complex at 7.

Manufacture of liposomes by isopropanol injection: characterization of the method.

Unilamellar liposomes are conventionally prepared by rapid injection of an ethanolic solution of lipids into an aqueous medium. The aim of the present study was to control, more efficiently, vesicle diameter by using an alternative solvent. The results show that isopropanol injection is a good alternative to ethanol injection for the manufacture of liposomes.

Two-dimensional crystallisation of soluble protein complexes.

This method aims at providing structural information on protein or nucleoprotein complexes by high-resolution electron microscopy. The objective is to promote the self-assembly of the macromolecules into two-dimensional crystals in order to use electron crystallography methods. When combined with observations in the frozen hydrated states and dedicated image processing software these methods can provide detailed 3-D models of the complex.

Mapping the initiator binding Taf2 subunit in the structure of hydrated yeast TFIID.

The general transcription factor TFIID is a large multisubunit complex required for the transcription of most protein-encoding genes by RNA polymerase II. Taking advantage of a TFIID preparation partially depleted in the initiator-binding Taf2p subunit, we determined the conformational and biochemical variations of the complex by electron tomography and cryo-electron microscopy of single molecules. Image analysis revealed the extent of conformational flexibility of the complex and the selection of the most homogeneous TFIID subpopulation allowed us to determine an improved structural model at 23 Angstroms resolution.

Filamentous condensation of DNA induced by pegylated poly-L-lysine and transfection efficiency.

In this paper we propose a detailed analysis of structural and morphological properties of two poly-L-lysine (PLL)-based transfection formulations, PLL/DNA and pegylated PLL (PLL-g-PEG)/DNA, by means of atomic force microscopy (AFM) and transmission electron microscopy (TEM). Comparing PLL-g-PEG/DNA with PLL/DNA polyplexes, we demonstrate that, due to the presence of PEG, the particles differ not only in size, shape, and crystalline structure, but also in transfection efficiency. While PLL condensates DNA in large agglomerates, PLL grafted with polyethylene glycol 2000 can condensate DNA in long filaments with diameters of some nanometers (6-20 nm).

Microstructural investigation of magnetic CoFe2O4 nanowires inside carbon nanotubes by electron tomography.

Magnetic nanowires of CoFe 2O4 were casted inside the channel of multiwall carbon nanotubes by mild chemical synthesis. A detailed investigation of these nanowires was performed using mainly the electron tomography technique; this study provides a complete characterization of their microstructure in terms of the spatial organization and the size distribution of individual particles forming the nanowire as well as its residual porosity. In particular, we have shown that the size of the CoFe 2O4 monocrystalline particles is closely dependent on the location of the particle within the nanotube, i.

Structure and oligomeric state of human transcription factor TFIIE.

The general RNA polymerase II transcription factor TFIIE, which is composed of two subunits, has essential roles in both transcription initiation and promoter escape. Electron microscopy analysis of negatively stained human TFIIE showed a large proportion of alpha/beta heterodimers as well as a small proportion of tetramers. Analytical ultracentrifugation, chemical crosslinking, pulldown experiments and cryo-electron microscopy confirmed that TFIIE is a alpha/beta heterodimer in solution.

Immobilization of biotinylated DNA on 2-D streptavidin crystals.

The structural study of transient nucleoprotein complexes by electron microscopy is hampered by the coexistence of multiple interaction states leading to an heterogeneous image population. To tackle this problem, we have investigated the controlled immobilization of double stranded DNA molecules and of nucleoprotein complexes onto a support suitable for cryo-electron microscopy observation. The DNA was end-labeled with a biotin moiety in order to decorate, or to be incorporated into, two-dimensional streptavidin crystals formed in contact of a biotinylated lipid layer.

Expression of FLAG fusion proteins in insect cells: application to the multi-subunit transcription/DNA repair factor TFIIH.

The multi-subunit transcription/DNA repair factor TFIIH was used as a model system to show that the expression of FLAG fusion proteins in insect cells constitutes a versatile tool for both structural and functional investigations. In the present study, we have constructed recombinant baculoviruses expressing the four core TFIIH subunits fused at their N-terminus to the FLAG peptide. Using these recombinant viruses we have established protocols based on anti-FLAG immunoaffinity chromatography that allow the systematic analysis of pairwise interaction within multiprotein complexes and have developed a double tag strategy (FLAG and hexahistidine tags) for the identification and purification of stable TFIIH subcomplexes.