PXD016241-2

PXD016241 is an original dataset announced via ProteomeXchange.

Title	Identifying nucleic acid-associated proteins in Mycobacterium smegmatis by mass spectrometry-based proteomics
Description	Background: The intracellular pathogen Mycobacterium tuberculosis is known to encounter several stress conditions during infection, including low pH, hypoxia, starvation and oxidative and nitrosative stress. Mycobacterial adaptation to stress conditions is, in part, mediated by several nucleic acid-associated proteins. In this study, we sought to establish an affinity purification-mass spectrometry (AP-MS) approach that would enable the collective identification of nucleic acid-associated proteins in mycobacteria. We hypothesized that targeting the RNA polymerase complex through affinity purification would allow for the identification of RNA- and DNA-associated proteins that not only maintain the bacterial chromosome but also enable transcription and translation. Results: AP-MS analysis of the RNA polymerase β-subunit cross-linked to nucleic acids identified 275 putative nucleic acid-associated proteins in the model organism Mycobacterium smegmatis under standard culturing conditions. The AP-MS approach successfully identified proteins that are known to make up the RNA polymerase complex, as well as several other known RNA polymerase complex-associated proteins such as a DNA polymerase, sigma factors, transcriptional regulators, and helicases. Gene ontology enrichment analysis of the identified proteins revealed that this approach selected for proteins with GO terms associated with nucleic acids and cellular metabolism. Importantly, we identified several proteins of unknown function not previously known to be associated with nucleic acids. Validation of several candidate nucleic acid-associated proteins demonstrated for the first time DNA association of ectopically expressed MSMEG_1060, MSMEG_2695 and MSMEG_4306 through affinity purification. Conclusions: Effective identification of nucleic acid-associated proteins, which make up the RNA polymerase complex as well as other DNA- and RNA-associated proteins, was facilitated by affinity purification of the RNA polymerase β-subunit in M. smegmatis. The successful identification of several transcriptional regulators suggest that our approach could be sensitive enough to investigate the nucleic acid-associated proteins that maintain cellular functions and mediate transcriptional and translational change in response to environmental stress.
HostingRepository	PRIDE
AnnounceDate	2020-03-05
AnnouncementXML	Submission_2020-04-16_22:07:40.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Nastassja Kriel
SpeciesList	scientific name: Mycolicibacterium smegmatis; NCBI TaxID: 1772;
ModificationList	No PTMs are included in the dataset
Instrument	Orbitrap Fusion

Revision	Datetime	Status	ChangeLog Entry
0	2019-11-12 02:44:43	ID requested
1	2020-03-05 00:25:24	announced
⏵ 2	2020-04-16 22:07:41	announced	2020-04-17: Updated publication reference for PubMed record(s): 32293251.
3	2024-10-22 04:59:35	announced	2024-10-22: Updated project metadata.

Kriel NL, Heunis T, Sampson SL, Gey van Pittius NC, Williams MJ, Warren RM, Identifying nucleic acid-associated proteins in Mycobacterium smegmatis by mass spectrometry-based proteomics. BMC Mol Cell Biol, 21(1):19(2020) [pubmed]

submitter keyword: RNA polymerase, Mycobacterium, Affinity-purification, nucleic acid-associated proteins

Robin Mark Warren
contact affiliation	Department of Biomedical Sciences, Stellenbosch University, South Africa
contact email	rw1@sun.ac.za
lab head
Nastassja Kriel
contact affiliation	Stellenbosch Univeristy
contact email	nastassja@sun.ac.za
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD016241
     1. Label: PRIDE project
     2. Name: Identifying nucleic acid-associated proteins in Mycobacterium smegmatis by mass spectrometry-based proteomics