A supraspliceosome model for large nuclear ribonucleoprotein particles based on mass determinations by scanning transmission electron microscopy

Pre-mRNA splicing is an important regulatory step in the expression of most eukaryotic genes. In vitro studies have shown splicing to occur within 50-60 S multi-component ribonucleoprotein (RNP) complexes termed spliceosomes. Studies of mammalian cell nuclei have revealed larger complexes that sediment at 200 S in sucrose gradients, termed large nuclear RNP (lnRNP) particles. These particles contain all factors required for pre-mRNA splicing, including the spliceosomal U snRNPs and protein splicing factors. Electron microscopy has shown them to consist of four apparently similar substructures. In this study, mass measurements by scanning transmission electron microscopy of freeze-dried mammalian lnRNP preparations, both confirm the similarity between the lnRNP particles and reveal the mass uniformity of their subunits. Thus, the tetrameric lnRNP particle has a mass of 21.1(+/-1.6) MDa, while each repeating subunit has a mass of 4.8(+/-0.5) MDa, which is close to the estimated mass of the fully assembled 60 S spliceosome. The 1.9 MDa discrepancy between the lnRNP particle's mass and the cumulative masses of its four subunits may be attributed to an additional domain frequently observed in the micrographs. Notably, strands and loops of RNA were often seen emanating from lnRNP particles positively stained with uranyl formate. Our results support the idea that the nuclear splicing machine is a supraspliceosome complex. For clarity, we define spliceosomes devoid of pre-mRNA as spliceosome cores, and propose that the supraspliceosome is constructed from one pre-mRNA, four spliceosome cores, each composed mainly of U snRNPs, and additional proteins. In this way a frame is provided to juxtapose exons about to be spliced

Three-dimensional reconstructions from cryo-electron microscopy images reveal an intimate complex between helicase DnaB and its loading partner DnaC

Background: DNA helicases play a fundamental role in all aspects of nucleic acid metabolism and defects in these enzymes have been implicated in a number of inherited human disorders. DnaB is the major replicative DNA helicase in Escherichia coli and has been used as a model system for studying the structure and function of hexameric helicases. The native protein is a hexamer of identical subunits, which in solution forms a complex with six molecules of the loading protein DnaC. DnaB is delivered from this complex onto the DNA template, with the subsequent release of DnaC. We report here the structures of the DnaB helicase hexamer and its complex with DnaC under a defined set of experimental conditions, as determined by three-dimensional cryoelectron microscopy. It was hoped that the structures would provide insight into the mechanisms of helicase activity. Results: The DnaB structure reveals that six DnaB monomers assemble as three asymmetric dimers to form a polar, ring-like hexamer. The hexamer has two faces, one displaying threefold and the other sixfold symmetry. The six DnaC protomers bind tightly to the sixfold face of the DnaB hexamer. This is the first report of a three-dimensional structure of a helicase obtained using cryoelectron microscopy, and the first report of the structure of a helicase in complex with a loading protein. Conclusions: The structures of the DnaB helicase and its complex with DnaC reveal some interesting structural features relevant to helicase function and to the assembly of the two-protein complex. The results presented here provide a basis for a more complete understanding of the structure and function of these important proteins

Domain organization of Mac-2 binding protein and its oligomerization to linear and ring-like structures

The multidomain Mac-2 binding protein (M2BP) is present in serum and in the extracellular matrix in the form of linear and ring-shaped oligomers, which interact with galectin-3, fibronectin, collagens, integrins and other large glycoproteins. Domain 1 of M2BP (M2BP-1) shows homology with the cysteine-rich SRCR domain of scavanger receptor. Domains 2 and 3 are related to the dimerization domains BTB/POZ and IVR of the Drosophila kelch protein. Recombinant M2BP, its N-terminal domain M2BP-1 and a fragment consisting of putative domains 2, 3 and 4 (M2BP-2,3,4) were investigated by scanning transmission electron microscopy, transmission electron microscopy, analytical ultracentrifugation and binding assays. The ring oligomers formed by the intact protein are comprised of approximately 14 nm long segments composed of two 92 kDa M2BP monomers. Although the rings vary in size, decamers predominate. The various linear oligomers also observed are probably ring precursors, dimers predominate. M2BP-1 exhibits a native fold, does not oligomerize and is inactive in cell attachment. M2BP-2,3,4 aggregates to heterogeneous, protein filled ring-like structures as shown by metal shadowed preparations. These aggregates retain the cell-adhesive potential indicating native folding. It is hypothesized that the rings provide an interaction pattern for multivalent interactions of M2BP with target molecules or complexes of ligands

Mitochondrial Lon of Saccharomyces cerevisiae is a ring-shaped protease with seven flexible subunits

Lon (or La) is a soluble, homooligomeric ATP-dependent protease. Mass determination and cryoelectron microscopy of pure mitochondrial Lon from Saccharomyces cerevisiae identify Lon as a flexible ring-shaped heptamer. In the presence of ATP or 5'-adenylylimidodiphosphate, most of the rings are symmetric and resemble other ATP-driven machines that mediate folding and degradation of proteins. In the absence of nucleotides, most of the rings are distorted, with two adjacent subunits forming leg-like protrusions. These results suggest that asymmetric conformational changes serve to power processive unfolding and translocation of substrates to the active site of the Lon protease

The reaction center complex from the green sulfur bacterium Chlorobium tepidum: A structural analysis by scanning transmission electron microscopy

The three-dimensional (3D) structure of the reaction center (RC) complex isolated from the green sulfur bacterium Chlorobium tepidum was determined from projections of negatively stained preparations by angular reconstitution. The purified complex contained the PscA, PscC, PscB, PscD subunits and the Fenna-Matthews-Olson (FMO) protein. Its mass was found to be 454 kDa by scanning transmission electron microscopy (STEM), indicating the presence of two copies of the PscA subunit, one copy of the PscB and PscD subunits, three FMO proteins and at least one copy of the PscC subunit. An additional mass peak at 183 kDa suggested that FMO trimers copurify with the RC complexes. Images of negatively stained RC complexes were recorded by STEM and aligned and classified by multivariate statistical analysis. Averages of the major classes indicated that different morphologies of the elongated particles (length = 19 nm, width = 8 nm) resulted from a rotation around the long axis. The 3D map reconstructed from these projections allowed visualization of the RC complex associated with one FMO trimer. A second FMO trimer could be correspondingly accommodated to yield a symmetric complex, a structure observed in a small number of side views and proposed to be the intact form of the RC complex. (C) 1999 Academic Press

Evaluating atomic models of F-actin with an undecagold-tagged phalloidin derivative

We have prepared an undecagold-tagged phalloidin derivative to determine this mushroom toxin's binding site and orientation within the F-actin filament by scanning transmission electron microscopy (STEM) and 3-D helical reconstruction. Remarkably, when stoichiometrically bound to F-actin, the undecagold moiety of the derivative could be directly visualized by STEM along the two half-staggered long-pitch helical strands of single filaments. Most importantly, the structural data obtained when combined with various biochemical constraints enabled us to critically evaluate two distinct atomic models of the F-actin filament (i.e. the Holmes-Lorenz versus the Schutt-Lindberg model). Taken together, our data are in excellent agreement with the Holmes-Lorenz model

The reaction center complex from the green sulfur bacterium Chlorobium tepidum: a structural analysis by scanning transmission electron microscopy

The three-dimensional (3D) structure of the reaction center (RC) complex isolated from the green sulfur bacterium Chlorobium tepidum was determined from projections of negatively stained preparations by angular reconstitution. The purified complex contained the PscA, PscC, PscB, PscD subunits and the Fenna-Matthews-Olson (FMO) protein. Its mass was found to be 454 kDa by scanning transmission electron microscopy (STEM), indicating the presence of two copies of the PscA subunit, one copy of the PscB and PscD subunits, three FMO proteins and at least one copy of the PscC subunit. An additional mass peak at 183 kDa suggested that FMO trimers copurify with the RC complexes. Images of negatively stained RC complexes were recorded by STEM and aligned and classified by multivariate statistical analysis. Averages of the major classes indicated that different morphologies of the elongated particles (length=19 nm, width=8 nm) resulted from a rotation around the long axis. The 3D map reconstructed from these projections allowed visualization of the RC complex associated with one FMO trimer. A second FMO trimer could be correspondingly accommodated to yield a symmetric complex, a structure observed in a small number of side views and proposed to be the intact form of the RC complex