Examining the complexity of human RNA polymerase complexes using HaloTag technology coupled to label free quantitative proteomics

Danette L. Daniels, Jacqui Méndez, Amber L. Mosley, Sreenivasa R. Ramisetty, Nancy Murphy, Hélène Benink, Keith V. Wood, Marjeta Urh, Michael P. Washburn

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

Efficient determination of protein interactions and cellular localization remains a challenge in higher order eukaryotes and creates a need for robust technologies for functional proteomics studies. To address this, the HaloTag technology was developed for highly efficient and rapid isolation of intracellular complexes and correlative in vivo cellular imaging. Here we demonstrate the strength of this technology by simultaneous capture of human eukaryotic RNA polymerases (RNAP) I, II, and III using a shared subunit, POLR2H, fused to the HaloTag. Affinity purifications showed successful isolation, as determined using quantitative proteomics, of all RNAP core subunits, even at expression levels near endogenous. Transient known RNAP II interacting partners were identified as well as three previously uncharacterized interactors. These interactions were validated and further functionally characterized using cellular imaging. The multiple capabilities of the HaloTag technology demonstrate the ability to efficiently isolate highly challenging multiprotein complexes, discover new interactions, and characterize cellular localization.

Original languageEnglish (US)
Pages (from-to)564-575
Number of pages12
JournalJournal of Proteome Research
Volume11
Issue number2
DOIs
StatePublished - Feb 3 2012

Keywords

  • cellular imaging
  • mass spectrometry
  • MudPIT
  • normalized spectral abundance factor
  • protein interactions
  • quantitative proteomics
  • RNA polymerases

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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  • Cite this

    Daniels, D. L., Méndez, J., Mosley, A. L., Ramisetty, S. R., Murphy, N., Benink, H., Wood, K. V., Urh, M., & Washburn, M. P. (2012). Examining the complexity of human RNA polymerase complexes using HaloTag technology coupled to label free quantitative proteomics. Journal of Proteome Research, 11(2), 564-575. https://doi.org/10.1021/pr200459c