Affinity purification of mammalian RNA polymerase I. Identification of an associated kinase.
Overlapping cDNA clones encoding the two largest subunits of rat RNA polymerase I, designated A194 and A127, were isolated from a Reuber hepatoma cDNA library. Analyses of the deduced amino acid sequences revealed that A194 and A127 are the homologues of yeast A190 and A135 and have homology to the beta' and beta subunits of Escherichia coli RNA polymerase I. Antibodies raised against the recombinant A194 and A127 proteins recognized single proteins of approximately 190 and 120 kDa on Western blots of total cellular proteins of mammalian origin. N1S1 cell lines expressing recombinant His-tagged A194 and FLAG-tagged A127 proteins were isolated. These proteins were incorporated into functional RNA polymerase I complexes, and active enzyme, containing FLAG-tagged A127, could be immunopurified to approximately 80% homogeneity in a single chromatographic step over an anti-FLAG affinity column. Immunoprecipitation of A194 from 32P metabolically labeled cells with anti-A194 antiserum demonstrated that this subunit is a phosphoprotein. Incubation of the FLAG affinity-purified RNA polymerase I complex with [gamma-32P]ATP resulted in autophosphorylation of the A194 subunit of RPI, indicating the presence of associated kinase(s). One of these kinases was demonstrated to be CK2, a serine/threonine protein kinase implicated in the regulation of cell growth and proliferation.
Hannan
R D
RD
Henry Hood Research Program, Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822-2618, USA.
Hempel
W M
WM
Cavanaugh
A
A
Arino
T
T
Dimitrov
S I
SI
Moss
T
T
Rothblum
L
L
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary; D008969:Molecular Sequence Data; D010766:Phosphorylation; D012318:RNA Polymerase I / Q000235:genetics / Q000302:isolation & purification*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D018076:DNA, Complementary; D011994:Recombinant Proteins; D012318:RNA Polymerase I