1,721,058 research outputs found
RNA polymerase II transcription compartments: from multivalent chromatin binding to liquid droplet formation?
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Dissecting the nascent human transcriptome by analysing the RNA content of transcription factories
No full textWhile mapping total and poly-adenylated human transcriptomes has now become routine, characterizing nascent transcripts remains challenging, largely because nascent RNAs have such short half-lives. Here, we describe a simple, fast and cost-effective method to isolate RNA associated with transcription factories, the sites responsible for the majority of nuclear transcription. Following stimulation of human endothelial cells with the pro-inflammatory cytokine TNFα, we isolate and analyse the RNA content of factories by sequencing. Comparison with total, poly(A)(+) and chromatin RNA fractions reveals that sequencing of purified factory RNA maps the complete nascent transcriptome; it is rich in intronic unprocessed transcript, as well as long intergenic non-coding (lincRNAs) and enhancer-associated RNAs (eRNAs), micro-RNA precursors and repeat-derived RNAs. Hence, we verify that transcription factories produce most nascent RNA and confer a regulatory role via their association with a set of specifically-retained non-coding transcripts
RippeLab/NSQFRAP: NSQFRAP_v1.1
No full textRelease associated with Trojanowski, Jorge; Frank, Lukas; Rademacher, Anne; Mücke, Norbert; Grigaitis, Pranas; Rippe, Karsten (2022), "Transcription activation is enhanced by multivalent interactions independent of phase separation." Unchanged compared to version 1.1Data storage at SDS@hd was funded by the Ministry of Science, Research and the Arts Baden-Württemberg (MWK) and the DFG through grants INST 35/1314-1 FUGG and INST 35/1503-1 FUGG
The GTPase cycle of the chloroplast import receptors Toc33/Toc34: implications from monomeric and dimeric structures
No full textTransport of precursor proteins across chloroplast membranes involves the GTPases Toc33/34 and Toc159 at the outer chloroplast envelope. The small GTPase Toc33/34 can homodimerize, but the regulation of this interaction has remained elusive. We show that dimerization is independent of nucleotide loading state, based on crystal structures of dimeric Pisum sativum Toc34 and monomeric Arabidopsis thaliana Toc33. An arginine residue is--in the dimer--positioned to resemble a GAP arginine finger. However, GTPase activation by dimerization is sparse and active site features do not explain catalysis, suggesting that the homodimer requires an additional factor as coGAP. Access to the catalytic center and an unusual switch I movement in the dimeric structure support this finding. Potential binding sites for interactions within the Toc translocon or with precursor proteins can be derived from the structures
Structure of a bacterial pyridoxal 5'-phosphate synthase complex
No full textVitamin B6 is an essential metabolic cofactor that has more functions in humans than any other single nutrient. Its de novo biosynthesis occurs through two mutually exclusive pathways that are absent in animals. The predominant pathway found in most prokaryotes, fungi, and plants has only recently been discovered. It is distinguished by a glutamine amidotransferase, which is remarkable in that it alone can synthesize the cofactor form, pyridoxal 5'-phosphate (PLP), directly from a triose and a pentose saccharide and glutamine. Here we report the 3D structure of the PLP synthase complex with substrate glutamine bound as well as those of the individual synthase and glutaminase subunits Pdx1 and Pdx2, respectively. The complex is made up of 24 protein units assembled like a cogwheel, a dodecameric Pdx1 to which 12 Pdx2 subunits attach. In contrast to the architecture of previously determined glutamine amidotransferases, macromolecular assembly is directed by an N-terminal alpha-helix on the synthase. Interaction with the synthase subunit leads to glutaminase activation, resulting in formation of an oxyanion hole, a prerequisite for catalysis. Mutagenesis permitted identification of the remote glutaminase and synthase catalytic centers and led us to propose a mechanism whereby ammonia shuttles between these active sites through a methionine-rich hydrophobic tunnel
X-ray crystal structure of Saccharomyces cerevisiae Pdx1 provides insights into the oligomeric nature of PLP synthases
No full textThe universal enzymatic cofactor vitamin B6 can be synthesized as pyridoxal 5-phosphate (PLP) by the glutamine amidotransferase Pdx1. We show that Saccharomyces cerevisiae Pdx1 is hexameric by analytical ultracentrifugation and by crystallographic 3D structure determination. Bacterial homologues were previously reported to exist in hexamer:dodecamer equilibrium. A small sequence insertion found in yeast Pdx1 elevates the dodecamer dissociation constant when introduced into Bacillus subtilis Pdx1. Further, we demonstrate that the yeast Pdx1 C-terminus contacts an adjacent subunit, and deletion of this segment decreases enzymatic activity 3.5-fold, suggesting a role in catalysis
Statistical and network-based methods for the analysis of chromatin accessibility maps in single cells
Full text linkIn questo lavoro, metodi provenienti dalla Fisica, dalla Statistica e dalla Teoria dei Grafi sono stati impiegati per caratterizzare ed analizzare profili di apertura e accessibilità della cromatina ottenuti con la tecnica ATAC-seq in singole cellule, nella fattispecie linfociti B provenienti da tre pazienti affetti da Leucemia Linfocitica Cronica.
Una pipeline bioinformatica è stata sviluppata per processare i dati di sequencing ed ottenere le posizioni accessibili del genoma per ciascuna cellula. La quantità di regioni aperte e la loro distribuzione spaziale lungo il DNA sono state caratterizzate. Infine, l’apertura simultanea nelle stesse singole cellule di regioni regolatrici è stata impiegata come metrica per valutare relazioni funzionali, e in questo modo grafi tra enhancer e promoter sono stati costruiti e le loro proprietà sono state analizzate.
La distribuzione spaziale lungo il genoma di regioni aperte consecutive ricapitola proprietà strutturali come gli array di nucleosomi e le strutture a loop della cromatina. Inoltre, i profili di accessibilità delle regioni regolatrici sono significativamente conservati nelle singole cellule. I network tra enhancer e promoter forniscono un modo per caratterizzare la rilevanza di ciascuna regione regolatrice in termini di centralità. Le statistiche sulla connettività tra enhancer e promoter confermano il modello di relazione uno-a-uno come il più frequente, in cui un promoter è regolato dall'enhancer ad esso più vicino. Infine, anche il funzionamento dei superenhancer è stato indagato.
In conclusione, ATAC-seq si rivela un'efficace tecnica per indagare l'apertura della cromatina in singole cellule, i cui profili di accessibilità ricapitolano caratteristiche strutturali e funzionali della cromatina. Al fine di indagare i meccanismi della malattia, il panorama di accessibilità dei lifociti tumorali può essere confrontato con quello di cellule sane e cellule trattate con farmaci epigenetici
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