ABSTRACT
Chromatin fibers must fold or coil in the process of chromosome condensation. Patterns of coiling have been demonstrated for reconstituted chromatin, but the actual trajectories of fibers in condensed states of chromosomes could not be visualized because of the high density of the material. We have exploited partial decondensation of mitotic chromosomes to reveal their internal structure at sub-nucleosomal resolution by cryo-electron tomography, without the use of stains, fixatives, milling, or sectioning. DNA gyres around nucleosomes were visible, allowing the nucleosomes to be identified and their orientations to be determined. Linker DNA regions were traced, revealing the trajectories of the chromatin fibers. The trajectories were irregular, with almost no evidence of coiling and no short- or long-range order of the chromosomal material. The 146-bp core particle, long known as a product of nuclease digestion, is identified as the native state of the nucleosome, with no regular spacing along the chromatin fibers.
Subject(s)
Chromosomes/ultrastructure , DNA/chemistry , Mitosis , Nucleosomes/metabolism , Amino Acid Motifs , Chromatin/chemistry , Cryoelectron Microscopy , Green Fluorescent Proteins/metabolism , HeLa Cells , Histones/chemistry , Humans , Microscopy, Fluorescence , Nucleosomes/chemistry , Spermidine/chemistry , TomographyABSTRACT
A computational method was developed to recover the three-dimensional coordinates of gold nanoparticles specifically attached to a protein complex from tilt-pair images collected by electron microscopy. The program was tested on a simulated dataset and applied to a real dataset comprising tilt-pair images recorded by cryo electron microscopy of RNA polymerase II in a complex with four gold-labeled single-chain antibody fragments. The positions of the gold nanoparticles were determined, and comparison of the coordinates among the tetrameric particles revealed the range of motion within the protein complexes.
Subject(s)
Gold/chemistry , Image Processing, Computer-Assisted/methods , Immunoglobulin Fragments , Metal Nanoparticles/chemistry , RNA Polymerase II , Cryoelectron Microscopy/methods , Immunoglobulin Fragments/chemistry , Immunoglobulin Fragments/metabolism , Models, Molecular , Protein Binding , RNA Polymerase II/chemistry , RNA Polymerase II/metabolismABSTRACT
The fibroblast growth factor FGF21 was labeled with molecularly defined gold nanoparticles (AuNPs), applied to human adipocytes, and imaged by cryo-electron tomography (cryo-ET). Most AuNPs were in pairs about 80 Å apart, on the outer cell surface. Pairs of AuNPs were also abundant inside the cells in clathrin-coated vesicles and endosomes. AuNPs were present but no longer paired in multivesicular bodies. FGF21 could thus be tracked along the endocytotic pathway. The methods developed here to visualize signaling coupled to endocytosis can be applied to a wide variety of cargo and may be extended to studies of other intracellular transactions.
Subject(s)
Cell Membrane/chemistry , Endocytosis/genetics , Endosomes/chemistry , Fibroblast Growth Factors/chemistry , Cell Movement/genetics , Clathrin-Coated Vesicles/chemistry , Clathrin-Coated Vesicles/metabolism , Electron Microscope Tomography , Fibroblast Growth Factors/isolation & purification , Gold/chemistry , Humans , Metal Nanoparticles/chemistry , Protein Transport/genetics , Signal Transduction , Surface PropertiesABSTRACT
Structure determination by transmission electron microscopy has revealed the long sought 144-gold atom particle. The structure exhibits deviations from face-centered cubic packing of the gold atoms, similar to the solution structure of another gold nanoparticle, and in contrast to a previous X-ray crystal structure. Evidence from analytical methods points to a low number of 3-mercaptobenzoic acid ligands covering the surface of the particle.
Subject(s)
Gold/chemistry , Metal Nanoparticles/chemistry , Microscopy, Electron, Scanning Transmission , Molecular Structure , Particle Size , Solubility , Surface Properties , Water/chemistryABSTRACT
By a modification of the method of Brust et al., water-soluble, thiolate-protected gold nanoparticles that are uniform in size were synthesized with no requirement for purification. The modification of the method was equilibration in the first step, which proved crucial for achieving size homogeneity. The thiol-to-gold ratio controlled the size of the particles, and the choice of thiol controlled the reactivity of the particles toward thiol exchange.
Subject(s)
Gold/chemistry , Metal Nanoparticles/chemistry , Sulfhydryl Compounds/chemistry , Ligands , Microscopy, Electron, Transmission/methods , Particle Size , Single-Chain Antibodies/chemistry , Solubility , Surface Properties , WaterABSTRACT
Structure determination of gold nanoparticles (AuNPs) is necessary for understanding their physical and chemical properties, but only one AuNP larger than 1 nanometer in diameter [a 102-gold atom NP (Au102NP)] has been solved to atomic resolution. Whereas the Au102NP structure was determined by x-ray crystallography, other large AuNPs have proved refractory to this approach. Here, we report the structure determination of a Au68NP at atomic resolution by aberration-corrected transmission electron microscopy, performed with the use of a minimal electron dose, an approach that should prove applicable to metal NPs in general. The structure of the Au68NP was supported by small-angle x-ray scattering and by comparison of observed infrared absorption spectra with calculations by density functional theory.
ABSTRACT
The protein density and arrangement of subunits of a complete, 32-protein, RNA polymerase II (pol II) transcription pre-initiation complex (PIC) were determined by means of cryogenic electron microscopy and a combination of chemical cross-linking and mass spectrometry. The PIC showed a marked division in two parts, one containing all the general transcription factors (GTFs) and the other pol II. Promoter DNA was associated only with the GTFs, suspended above the pol II cleft and not in contact with pol II. This structural principle of the PIC underlies its conversion to a transcriptionally active state; the PIC is poised for the formation of a transcription bubble and descent of the DNA into the pol II cleft.
Subject(s)
Gene Expression Regulation, Fungal , Multiprotein Complexes/chemistry , RNA Polymerase II/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/enzymology , Transcription Factors, General/chemistry , Transcription Initiation, Genetic , Cryoelectron Microscopy , DNA, Fungal/chemistry , DNA, Fungal/genetics , Nucleic Acid Conformation , Protein Conformation , Saccharomyces cerevisiae/geneticsABSTRACT
Structures of complete 10-subunit yeast TFIIH and of a nested set of subcomplexes, containing 5, 6, and 7 subunits, have been determined by electron microscopy (EM) and 3D reconstruction. Consistency among all the structures establishes the location of the "minimal core" subunits (Ssl1, Tfb1, Tfb2, Tfb4, and Tfb5), and additional densities can be specifically attributed to Rad3, Ssl2, and the TFIIK trimer. These results can be further interpreted by placement of previous X-ray structures into the additional densities to give a preliminary picture of the RNA polymerase II preinitiation complex. In this picture, the key catalytic components of TFIIH, the Ssl2 ATPase/helicase and the Kin28 protein kinase are in proximity to their targets, downstream promoter DNA and the RNA polymerase C-terminal domain.
Subject(s)
Protein Subunits/chemistry , Saccharomyces cerevisiae/metabolism , Transcription Factor TFIIH/chemistry , Calmodulin/metabolism , Electrophoresis, Polyacrylamide Gel , Models, Molecular , Multiprotein Complexes/isolation & purification , Staining and Labeling , Transcription Factor TFIIH/isolation & purification , Transcription Factor TFIIH/ultrastructureABSTRACT
Most eukaryotic pre-mRNAs contain non-coding sequences (introns) that must be removed in order to accurately place the coding sequences (exons) in the correct reading frame. This critical regulatory pre-mRNA splicing event is fundamental in development and cancer. It occurs within a mega-Dalton multicomponent machine composed of RNA and proteins, which undergoes dynamic changes in RNA-RNA, RNA-protein, and protein-protein interactions during the splicing reaction. Recent years have seen progress in functional and structural analyses of the splicing machine and its subcomponents, and this review is focused on structural aspects of the pre-mRNA splicing machine and their mechanistic implications on the splicing of multi-intronic pre-mRNAs. It brings together, in a comparative manner, structural information on spliceosomes and their intermediates in the stepwise assembly process in vitro, and on the preformed supraspliceosomes, which are isolated from living cell nuclei, with a view of portraying a consistent picture.
Subject(s)
RNA Precursors/genetics , RNA Splicing/genetics , Cryoelectron Microscopy , Image Processing, Computer-Assisted , Models, Molecular , RNA Precursors/ultrastructure , RNA, Fungal/genetics , RNA, Fungal/ultrastructure , Schizosaccharomyces/genetics , Spliceosomes/genetics , Spliceosomes/physiology , Spliceosomes/ultrastructureABSTRACT
Regulation of eukaryotic gene expression is achieved at different levels, which require accurate coordination. Macromolecular assemblies that exist as pre-formed entities can account for such coordination. Processing of pre-mRNA represents one step in this cascade of regulatory events but, moreover, provides explanation for protein versatility. The cellular machine where splicing of pre-mRNA, as well as additional processing events, take place in vivo is termed the supraspliceosome. Here, we show that the supraspliceosome is composed of four active spliceosomes, termed native spliceosomes, connected to each other by the pre-mRNA. Cleavage of pre-mRNA shows that its integrity is essential for the stability of the supraspliceosome. Furthermore, supraspliceosomes can be reconstituted in vitro, from purified native spliceosomes by addition of synthetic pre-mRNAs, providing further support to the supraspliceosome as a preassembled biological complex. The internal setting of the native spliceosomes within the supraspliceosome is most suitable to enable the communication between these structures, which is crucial in order to achieve regulated splicing.
Subject(s)
RNA Precursors/metabolism , RNA Processing, Post-Transcriptional , Spliceosomes/metabolism , HeLa Cells , Humans , Magnesium/metabolism , Models, Molecular , Multiprotein Complexes , Ribonuclease H/metabolism , Spliceosomes/ultrastructureABSTRACT
Splicing of pre-mRNA occurs in a multicomponent macromolecular machine--the spliceosome. The spliceosome can be assembled in vitro by a stepwise assembly of a number of snRNPs and additional proteins on exogenously added pre-mRNA. In contrast, splicing in vivo occurs in preformed particles where endogenous pre-mRNAs are packaged with all five spliceosomal U snRNPs (penta-snRNP) together with other splicing factors. Here we present a three-dimensional image reconstruction by cryo-electron microscopy of native spliceosomes, derived from cell nuclei, at a resolution of 20 angstroms. The structure revealed an elongated globular particle made up of two distinct subunits connected to each other leaving a tunnel in between. We show here that the larger subunit is a suitable candidate to accommodate the penta-snRNP, and that the tunnel could accommodate the pre-mRNA component of the spliceosome. The features this structure reveals provide new insight into the global architecture of the native splicing machine.
