Browse
Submit Data
Databases
API
Help

Dataset Information

32 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Analyzing RNA polymerase III by electron cryomicroscopy.

ABSTRACT: Recent electron cryomicroscopy reconstructions have provided new insights into the overall organization of yeast RNA polymerase (Pol) III, responsible for the synthesis of small, non-translated RNAs. The structure of the free Pol III enzyme at 10 Å resolution provides an accurate framework to better understand its overall architecture and the structural organization and functional role of two Pol III-specific subcomplexes. Cryo-EM structures of elongating Pol III bound to DNA/RNA scaffolds show the rearrangement of the Pol III-specific subcomplexes that enclose incoming DNA. In one reconstruction downstream DNA and newly transcribed RNA can be followed over considerably longer distances as in the crystal structure of elongating Pol II. The Pol III transcription machinery is increasingly recognized as a possible target for cancer therapy. The recent cryo-EM reconstructions contribute to the molecular understanding of Pol III transcription as a prerequisite for targeting its components.

SUBMITTER: Fernandez-Tornero C

PROVIDER: S-EPMC3256353 | biostudies-literature | 2011 Sep-Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Analyzing RNA polymerase III by electron cryomicroscopy.

Fernández-Tornero Carlos C Böttcher Bettina B Rashid Umar Jan UJ Müller Christoph W CW

RNA biology 20110901 5

Recent electron cryomicroscopy reconstructions have provided new insights into the overall organization of yeast RNA polymerase (Pol) III, responsible for the synthesis of small, non-translated RNAs. The structure of the free Pol III enzyme at 10 Å resolution provides an accurate framework to better understand its overall architecture and the structural organization and functional role of two Pol III-specific subcomplexes. Cryo-EM structures of elongating Pol III bound to DNA/RNA scaffolds show ...[more]

PMID: 21881405

Similar Datasets

Ex vivo visualization of RNA polymerase III-specific gene activity with electron microscopy.

Project description:The direct study of transcription or DNA-protein-binding events, requires imaging of individual genes at molecular resolution. Electron microscopy (EM) can show local detail of the genome. However, direct visualization and analysis of specific individual genes is currently not feasible as they cannot be unambiguously localized in the crowded, landmark-free environment of the nucleus. Here, we present a method for the genomic insertion of gene clusters that can be localized and imaged together with their associated protein complexes in the EM. The method uses CRISPR/Cas9 technology to incorporate several genes of interest near the 35S rRNA gene, which is a frequently occurring, easy-to-identify genomic locus within the nucleolus that can be used as a landmark in micrographs. As a proof of principle, we demonstrate the incorporation of the locus-native gene RDN5 and the locus-foreign gene HSX1. This led to a greater than 7-fold enrichment of RNA polymerase III (Pol III) complexes associated with the genes within the field of view, allowing for a significant increase in the analysis yield. This method thereby allows for the insertion and direct visualization of gene clusters for a range of analyses, such as changes in gene activity upon alteration of cellular or external factors.

| S-EPMC7895982 | biostudies-literature

Charge accumulation in electron cryomicroscopy.

Project description:When irradiated in a transmission electron microscope, plunge-frozen, amorphous water ice specimens accumulate a pattern of static charge that changes dynamically as the specimen is irradiated, and which can deflect the transmitted electrons and blur the resultant micrographs. Here we provide a physical description of this charge accumulation and characterise its dynamic behaviour in the context of low-dose electron cryomicroscopy (cryoEM). We observe the accumulation of positive charge in the primary irradiation area as expected from earlier work. To our surprise, we also observed a build-up of negative charge in nearby unirradiated regions of the specimen. Using a standard carbon support foil containing a pure water ice specimen, we collect a portion of this negative charge in the micrometer sized specimen holes which act as electrostatic lenses. These unusual, diverging micro-lenses are extremely sensitive charge detectors that allow us to directly measure the magnitude and dynamics of charge accumulation and neutralisation that occur during cryoEM imaging. Using these measurements, we find that the build-up of charge on the specimen saturates to a dynamic equilibrium at an electron fluence which is orders of magnitude lower than required for a typical low-dose micrograph. The measurements here will guide the development of optimal imaging conditions for biological specimens and contribute to a complete theory of information loss in electron cryomicroscopy.

| S-EPMC5862658 | biostudies-literature

Electron microscopy: Ultrastable gold substrates for electron cryomicroscopy.

Project description:Despite recent advances, the structures of many proteins cannot be determined by electron cryomicroscopy because the individual proteins move during irradiation. This blurs the images so that they cannot be aligned with each other to calculate a three-dimensional density. Much of this movement stems from instabilities in the carbon substrates used to support frozen samples in the microscope. Here we demonstrate a gold specimen support that nearly eliminates substrate motion during irradiation. This increases the subnanometer image contrast such that α helices of individual proteins are resolved. With this improvement, we determine the structure of apoferritin, a smooth octahedral shell of α-helical subunits that is particularly difficult to solve by electron microscopy. This advance in substrate design will enable the solution of currently intractable protein structures.

| S-EPMC4296556 | biostudies-literature

Multifunctional graphene supports for electron cryomicroscopy.

Project description:With recent technological advances, the atomic resolution structure of any purified biomolecular complex can, in principle, be determined by single-particle electron cryomicroscopy (cryoEM). In practice, the primary barrier to structure determination is the preparation of a frozen specimen suitable for high-resolution imaging. To address this, we present a multifunctional specimen support for cryoEM, comprising large-crystal monolayer graphene suspended across the surface of an ultrastable gold specimen support. Using a low-energy plasma surface modification system, we tune the surface of this support to the specimen by patterning a range of covalent functionalizations across the graphene layer on a single grid. This support design reduces specimen movement during imaging, improves image quality, and allows high-resolution structure determination with a minimum of material and data.

| S-EPMC6575631 | biostudies-literature

RNA polymerase III interactome

Project description:Many regulatory proteins and complexes have been identified which influence transcription by RNA polymerase (pol) II with a fine precision. In comparison, only a few regulatory proteins are known for pol III, which transcribes mostly house-keeping and non-coding RNAs. Yet, pol III transcription is precisely regulated under various stress conditions like starvation. We used proteomic approaches and pol III transcription complex components TFIIIC (Tfc6), pol III (Rpc128) and TFIIIB (Brf1) as baits to find identify the potential interactors through mass spectrometry-based proteomics. A large number of proteins were found in the interactome, which includes known chromatin modifiers, factors and regulators of transcription by pol I and pol II.

2019-01-02 | PXD003748 | Pride

Characterization of human RNA polymerase III identifies orthologues for Saccharomyces cerevisiae RNA polymerase III subunits.

Project description:Unlike Saccharomyces cerevisiae RNA polymerase III, human RNA polymerase III has not been entirely characterized. Orthologues of the yeast RNA polymerase III subunits C128 and C37 remain unidentified, and for many of the other subunits, the available information is limited to database sequences with various degrees of similarity to the yeast subunits. We have purified an RNA polymerase III complex and identified its components. We found that two RNA polymerase III subunits, referred to as RPC8 and RPC9, displayed sequence similarity to the RNA polymerase II RPB7 and RPB4 subunits, respectively. RPC8 and RPC9 associated with each other, paralleling the association of the RNA polymerase II subunits, and were thus paralogues of RPB7 and RPB4. Furthermore, the complex contained a prominent 80-kDa polypeptide, which we called RPC5 and which corresponded to the human orthologue of the yeast C37 subunit despite limited sequence similarity. RPC5 associated with RPC53, the human orthologue of S. cerevisiae C53, paralleling the association of the S. cerevisiae C37 and C53 subunits, and was required for transcription from the type 2 VAI and type 3 human U6 promoters. Our results provide a characterization of human RNA polymerase III and show that the RPC5 subunit is essential for transcription.

| S-EPMC134740 | biostudies-literature

Computational Methods for Single-Particle Electron Cryomicroscopy.

Project description:Single-particle electron cryomicroscopy (cryo-EM) is an increasingly popular technique for elucidating the three-dimensional structure of proteins and other biologically significant complexes at near-atomic resolution. It is an imaging method that does not require crystallization and can capture molecules in their native states. In single-particle cryo-EM, the three-dimensional molecular structure needs to be determined from many noisy two-dimensional tomographic projections of individual molecules, whose orientations and positions are unknown. The high level of noise and the unknown pose parameters are two key elements that make reconstruction a challenging computational problem. Even more challenging is the inference of structural variability and flexible motions when the individual molecules being imaged are in different conformational states. This review discusses computational methods for structure determination by single-particle cryo-EM and their guiding principles from statistical inference, machine learning, and signal processing that also play a significant role in many other data science applications.

| S-EPMC8412055 | biostudies-literature

Autoregulation of an RNA polymerase II promoter by the RNA polymerase III transcription factor III C (TF(III)C) complex.

Project description:Extra TF(III)C (ETC) sites are chromosomal locations bound in vivo by the RNA polymerase III (Pol III) transcription factor III C (TF(III)C) complex, but are not necessarily associated with Pol III transcription. Although the location of ETC sequences are conserved in budding yeast, and similar sites are found in other organisms, their functions are largely unstudied. One such site, ETC6 in Saccharomyces cerevisiae, lies upstream of TFC6, a gene encoding a subunit of the TF(III)C complex itself. Promoter analysis shows that the ETC6 B-box sequence is involved in autoregulation of the TFC6 promoter. Mutation of ETC6 increases TFC6 mRNA levels, whereas mutation immediately upstream severely weakens promoter activity. A temperature-sensitive mutation in TFC3 that weakens DNA binding of TF(III)C also results in increased TFC6 mRNA levels; however, no increase is observed in mutants of TF(III)B or Pol III subunits, demonstrating a specific role for the TF(III)C complex in TFC6 promoter regulation. Chromatin immunoprecipitation shows an inverse relationship of TF(III)C occupancy at ETC6 versus TFC6 mRNA levels. Overexpression of TFC6 increases association of TF(III)C at ETC6 (and other loci) and results in reduced expression of a TFC6 promoter-URA3 reporter gene. Both of these effects are dependent on the ETC6 B-box. These results demonstrate that the TFC6 promoter is directly regulated by the TF(III)C complex, a demonstration of an RNA polymerase II promoter being directly responsive to a core Pol III transcription factor complex. This regulation could have implications in controlling global tRNA expression levels.

| S-EPMC3100981 | biostudies-literature

Extragenic accumulation of RNA polymerase II enhances transcription by RNA polymerase III.

Project description:Recent genomic data indicate that RNA polymerase II (Pol II) function extends beyond conventional transcription of primarily protein-coding genes. Among the five snRNAs required for pre-mRNA splicing, only the U6 snRNA is synthesized by RNA polymerase III (Pol III). Here we address the question of how Pol II coordinates the expression of spliceosome components, including U6. We used chromatin immunoprecipitation (ChIP) and high-resolution mapping by PCR to localize both Pol II and Pol III to snRNA gene regions. We report the surprising finding that Pol II is highly concentrated approximately 300 bp upstream of all five active human U6 genes in vivo. The U6 snRNA, an essential component of the spliceosome, is synthesized by Pol III, whereas all other spliceosomal snRNAs are Pol II transcripts. Accordingly, U6 transcripts were terminated in a Pol III-specific manner, and Pol III localized to the transcribed gene regions. However, synthesis of both U6 and U2 snRNAs was alpha-amanitin-sensitive, indicating a requirement for Pol II activity in the expression of both snRNAs. Moreover, both Pol II and histone tail acetylation marks were lost from U6 promoters upon alpha-amanitin treatment. The results indicate that Pol II is concentrated at specific genomic regions from which it can regulate Pol III activity by a general mechanism. Consequently, Pol II coordinates expression of all RNA and protein components of the spliceosome.

| S-EPMC2082468 | biostudies-literature

Structure of human RNA polymerase III.

Project description:In eukaryotes, RNA Polymerase (Pol) III is specialized for the transcription of tRNAs and other short, untranslated RNAs. Pol III is a determinant of cellular growth and lifespan across eukaryotes. Upregulation of Pol III transcription is observed in cancer and causative Pol III mutations have been described in neurodevelopmental disorders and hypersensitivity to viral infection. Here, we report a cryo-EM reconstruction at 4.0 Å of human Pol III, allowing mapping and rationalization of reported genetic mutations. Mutations causing neurodevelopmental defects cluster in hotspots affecting Pol III stability and/or biogenesis, whereas mutations affecting viral sensing are located in proximity to DNA binding regions, suggesting an impairment of Pol III cytosolic viral DNA-sensing. Integrating x-ray crystallography and SAXS, we also describe the structure of the higher eukaryote specific RPC5 C-terminal extension. Surprisingly, experiments in living cells highlight a role for this module in the assembly and stability of human Pol III.

| S-EPMC7747717 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data