E1BF is an essential RNA polymerase I transcription factor with an intrinsic protein kinase activity that can modulate rRNA gene transcription.
We previously described the purification and characterization of E1BF, a rat rRNA gene core promoter-binding factor that consists of two polypeptides of 89 and 79 kDa. When this factor was incubated in the absence of any exogenous protein kinase under conditions optimal for protein phosphorylation, the 79-kDa polypeptide of E1BF was selectively phosphorylated. The labeled phosphate could be removed from the E1BF polypeptide by treatment with calf intestinal alkaline phosphatase or potato acid phosphatase. Elution of the protein from the E1BF-promoter complex formed in an electrophoretic mobility-shift assay followed by incubation of the concentrated eluent with [gamma-32P] ATP resulted in the selective labeling of the 79-kDa band. The E1BF-associated protein kinase did not phosphorylate casein or histone H1. Fraction DE-B, a preparation containing RNA polymerase I and all polymerase I transcription factors (including E1BF), lost polymerase I transcriptional activity when treated with phosphatase. The phosphatase-induced inactivation of polymerase I activity associated with fraction DE-B could be reversed by the addition of purified E1BF. Treatment of purified E1BF with heat, SDS, or an ATP affinity analog eliminated its capacity to reactivate dephosphorylated fraction DE-B. These data demonstrate that (i) polymerase I promoter-binding factor E1BF contains an intrinsic substrate-specific protein kinase and (ii) E1BF is an essential polymerase I transcription factor that can modulate rRNA gene transcription by protein phosphorylation. Further, these studies have provided a direct means to identify a protein kinase or any other enzyme that can interact with a specific DNA sequence.
Department of Pharmacology and Molecular Biology, Chicago Medical School, IL 60064.
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D000818:Animals; D034961:Antigens, Nuclear; D004265:DNA Helicases; D004275:DNA, Ribosomal / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000502:physiology*; D005786:Gene Expression Regulation; D066298:In Vitro Techniques; D000072200:Ku Autoantigen; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000502:physiology*; D010750:Phosphoproteins / Q000502:physiology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000502:physiology*; D012318:RNA Polymerase I / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000235:genetics*; D051381:Rats; D013379:Substrate Specificity; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic
D034961:Antigens, Nuclear; D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D012335:RNA, Ribosomal; D014157:Transcription Factors; D011494:Protein Kinases; D012318:RNA Polymerase I; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; C000606511:Xrcc6 protein, rat; D000072200:Ku Autoantigen