title: Examining the complexity of human RNA polymerase complexes using HaloTag technology coupled to label free quantitative proteomics.
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.
authors.affiliation: Promega Corporation , 2800 Woods Hollow Road, Madison, Wisconsin 53711, United States. danette.daniels@promega.com
authors.forename: Danette L
authors.initials: DL
authors.lastname: Daniels
authors.forename: Jacqui
authors.initials: J
authors.lastname: Méndez
authors.forename: Amber L
authors.initials: AL
authors.lastname: Mosley
authors.forename: Sreenivasa R
authors.initials: SR
authors.lastname: Ramisetty
authors.forename: Nancy
authors.initials: N
authors.lastname: Murphy
authors.forename: Hélène
authors.initials: H
authors.lastname: Benink
authors.forename: Keith V
authors.initials: KV
authors.lastname: Wood
authors.forename: Marjeta
authors.initials: M
authors.lastname: Urh
authors.forename: Michael P
authors.initials: MP
authors.lastname: Washburn
mesh_terms: D002460:Cell Line; D002467:Cell Nucleus; D019295:Computational Biology; D003593:Cytoplasm; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism; D030562:Databases, Protein; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D015335:Molecular Probes / Q000737:chemistry* / Q000378:metabolism; D046912:Multiprotein Complexes; D021122:Protein Subunits / Q000032:analysis*; D040901:Proteomics / Q000379:methods*
chemical_list: D015335:Molecular Probes; D046912:Multiprotein Complexes; D021122:Protein Subunits; D012321:DNA-Directed RNA Polymerases