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Identifying nucleic acid-associated proteins in Mycobacterium smegmatis by mass spectrometry-based proteomics

Lookup NU author(s): Dr Tiaan Heunis



This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


© 2020 The Author(s). Background: Transcriptional responses required to maintain cellular homeostasis or to adapt to environmental stress, 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.

Publication metadata

Author(s): Kriel NL, Heunis T, Sampson SL, Gey Van Pittius NC, Williams MJ, Warren RM

Publication type: Article

Publication status: Published

Journal: BMC Molecular and Cell Biology

Year: 2020

Volume: 21

Online publication date: 23/03/2020

Acceptance date: 09/03/2020

Date deposited: 06/04/2020

ISSN (electronic): 2661-8850

Publisher: BioMed Central Ltd


DOI: 10.1186/s12860-020-00261-6


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