Identifying nucleic acid-associated proteins in Mycobacterium smegmatis by mass spectrometry-based proteomics
Date
2020-03-23
Journal Title
Journal ISSN
Volume Title
Publisher
BioMed Central
Abstract
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 acidassociated
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.
Description
CITATION: Kriel, N. L., et al. 2020. Identifying nucleic acid-associated proteins in Mycobacterium smegmatis by mass spectrometry-based proteomics. BMC Molecular and Cell Biology, 21:19, doi:10.1186/s12860-020-00261-6.
The original publication is available at https://bmcmolcellbiol.biomedcentral.com
The original publication is available at https://bmcmolcellbiol.biomedcentral.com
Keywords
Mycobacterium tuberculosis -- Bactria, Nucleoproteins -- Identification, Mass spectrometry, Proteomics, RNA polymerases
Citation
Kriel, N. L., et al. 2020. Identifying nucleic acid-associated proteins in Mycobacterium smegmatis by mass spectrometry-based proteomics. BMC Molecular and Cell Biology, 21:19, doi:10.1186/s12860-020-00261-6