Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

In-cell architecture of an active transcription-translation complex

ABSTRACT: Structural biology performed inside cells can capture molecular machines in action within their native context. Here we develop an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae. We combine whole-cell crosslinking mass spectrometry, cellular cryo-electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at sub-nanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpoint NusA at the interface between the ribosome and a NusG-bound elongating RNAP, and propose it could mediate transcription-translation coupling. Transcription inhibition stalls and rearranges the expressome, whereas translation inhibition dissociates it, demonstrating that the elongating expressome architecture requires active translation and transcription within the cell.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mycoplasma Pneumoniae (strain Atcc 29342 / M129)

SUBMITTER: Francis O'Reilly

LAB HEAD: Juri Rappsilber

PROVIDER: PXD017711 | Pride | 2020-07-30

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Similar Datasets

In-cell architecture of an active transcription-translation complex

Project description:Structural biology performed inside cells can capture molecular machines in action within their native context. Here we develop an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae. We combine whole-cell crosslinking mass spectrometry, cellular cryo-electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at sub-nanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpoint NusA at the interface between the ribosome and a NusG-bound elongating RNAP, and propose it could mediate transcription-translation coupling. Transcription inhibition stalls and rearranges the expressome, whereas translation inhibition dissociates it, demonstrating that the elongating expressome architecture requires active translation and transcription within the cell.

2020-07-30 | PXD017695 | Pride

Expression data from mouse limb tendon cells during development.

Project description:We have undertaken a screen of mouse limb tendon cells in order to identify molecular pathways involved in tendon development. Mouse limb tendon cells were isolated based on Scleraxis (Scx) expression at different stages of development: E11.5, E12.5 and E14.5 Microarray comparisons were carried out between tendon progenitor and differentiated stages. Forelimbs from E11.5, E12.5 and E14.5 Scx-GFP embryos were collected and dissociated with trypsin to obtain cell suspensions. Scx-positive tendon cells were isolated by FACS. RNA was extracted and Fragmented biotin-labelled cRNA samples were hybridized on Affymetrix Gene Chip Mouse Genome 430 2.0 arrays.

2014-01-18 | E-GEOD-54207 | biostudies-arrayexpress

Dynamic profiling of the protein life cycle in response to pathogens (protein)

Project description:Protein expression is regulated by production and degradation of mRNAs and proteins, but their specific relationships remain unknown. We combine measurements of protein production and degradation and mRNA dynamics to build a quantitative genomic model of the differential regulation of gene expression in LPS stimulated mouse dendritic cells. Changes in mRNA abundance play a dominant role in determining most dynamic fold changes in protein levels. Conversely, the preexisting proteome of proteins performing basic cellular functions is remodeled primarily through changes in protein production or degradation, accounting for over half of the absolute change in protein molecules in the cell. Thus, the proteome is regulated by transcriptional induction of novel cellular functions and remodeling of preexisting functions through the protein life cycle. Mouse primary dendritic cells were treated with LPS or mock stimulus and profiled over a 12-hour time course. Cells were grown in M-labeled SILAC media, which was replaced with H-labeled SILAC media at time 0. Aliquots were taken at 0, 0.5, 1, 2, 3, 4, 5, 6, 9, and 12 hours post-stimulation and added to equal volumes of a master mix of unlabeled (L) cells for the purpose of normalization. RNA-Seq was performed at 0, 1, 2, 4, 6, 9, and 12 hours post-stimulation.

2015-03-06 | E-GEOD-59792 | biostudies-arrayexpress

mir-19 and mir-92 regulate lipid metabolism and energy balance

Project description:Profile gene expression change in 3T3 L1 adipocytes after overexpression of microRNA mir-17/18a, mir-18a/19a/20a, and mir-19b/92a

2015-06-19 | E-MTAB-2662 | biostudies-arrayexpress

Enhancer activation during EGF response

Project description:We investigated the acetylation of H3K27, in normal condition and after stimulation of EGF and before or after the depletion of INTS11. Additionally, we examined transcription by sequencing the chromatin-bound fraction of RNA and by fractionating total RNA into polyadenylated and non-polyadenylated compartement. Comparison of the genome wide profiles of H3K27ac and RNA in HeLa cells

2015-08-25 | E-GEOD-68401 | biostudies-arrayexpress

Cross-linking mass spectrometry uncovers interactions between high-density lipoproteins and the SARS-CoV-2 spike glycoprotein

Project description:Multiple studies to date have shown that high-density lipoprotein (HDL) levels are reduced in patients with coronavirus disease 2019 (COVID-19), and that the extent of this reduction is associated with poor clinical outcomes. While lipoproteins are known to play a key role during the lifecycle of hepatitis C virus, the direct influence they have upon coronavirus (CoV) infections remains poorly understood. In this study we utilise cross-linking mass spectrometry (XL-MS) to determine circulating protein interactors of the severe acute respiratory syndrome (SARS)-CoV-2 spike glycoprotein. Spike was revealed to interact with HDL, through direct interactions with apolipoprotein (Apo)-A1, ApoC3 and ApoD, highlighting multiple modalities of how SARS-CoV-2 and HDL may interact. XL-MS of plasma isolated from COVID-19 patients uncovered HDL protein interaction networks, dominated by acute phase serum amyloid proteins (SAA), whereby serum amyloid A2 (SAA2) was shown to bind to ApoD. ApoD was confirmed to also interact with core apolipoproteins of both LDL and VLDL, suggesting that SARS-CoV-2 may bind multiple lipoprotein species. The interaction between ApoD and spike was further validated in cells using immunoprecipitation-MS, which uncovered a novel interaction between both ApoD and spike with membrane-associated progesterone receptor component 1 (PGRMC1). Mechanistically, XL-MS coupled with data-driven structural modelling determined that ApoD may interact within the receptor-binding domain (RBD) of spike, posing the hypothesis that ApoD may interfere with binding to the angiotensin-converting enzyme 2 (ACE2) receptor. However, utilising multiple cell-based assays we determined ApoD to have no effect upon viral replication or infectivity.

2023-07-20 | PXD041831 | Pride

Cross-linking mass spectrometry uncovers interactions between high-density lipoproteins and the SARS-CoV-2 spike glycoprotein

2023-07-20 | PXD042625 | Pride

Cross-linking mass spectrometry uncovers interactions between high-density lipoproteins and the SARS-CoV-2 spike glycoprotein

2023-07-20 | PXD036640 | Pride

Cross-linking mass spectrometry uncovers interactions between high-density lipoproteins and the SARS-CoV-2 spike glycoprotein

2023-07-20 | PXD041438 | Pride

Expanding the depth and sensitivity of cross-link identification by differential ion mobility using FAIMS

Project description:In cross-linking mass spectrometry (XL-MS), the depth and sensitivity of cross-link detection is often limited by the low abundance of cross-links compared to non-cross-linked peptides in the digestion mixture. To improve the identification efficiency of cross-links, here we present a gas-phase separation strategy using high field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to the Orbitrap Tribrid mass spectrometers. By enabling an additional peptide separation step in gas phase using the FAIMS device, we increase the number of cross-link identification by 22% for a medium complex sample and 59% for strong cation exchange-fractionated HEK293T cell lysate in XL-MS experiments using disuccinimidyl sulfoxide (DSSO) cross-linker. When disuccinimidyl suberate (DSS) cross-linker is in use, we are able to boost cross-link identification by 89% for the medium and 100% for the highly complex sample comparing to the analyses without FAIMS . Furthermore, we show that for medium complex samples, FAIMS enables the collection of single-shot XL-MS data with comparable depth to the corresponding sample fractionated by chromatography-based approaches. Altogether, we demonstrate FAIMS is highly beneficial for XL-MS studies by expanding the proteome coverage of cross-links while improving the efficiency and confidence of cross-link identification.

2020-07-22 | PXD019926 | Pride

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