Copurification of RNA polymerase I and the glucocorticoid-regulated transcription factor IC.
A simple and rapid method to copurify RNA polymerase I and the glucocorticoid-regulated transcription factor, TFIC, is described. This protocol results in 1262-fold purification and 15% total recovery of the enzyme and factors needed to support faithful transcription in vitro from cloned mouse rRNA gene (rDNA). Using this method, proteins involved in rDNA transcription were purified from exponentially growing lymphosarcoma P1798 cells as well as cells treated with 0.1 microM dexamethasone. A combination of transcription and reconstitution assays using G-free cassette-containing constructs and polyacrylamide gel electrophoretic analysis upon silver staining were used to detect TFIC activity as well as the characteristic TFIC polypeptides in control and dexamethasone-treated cell extracts. Treatment of P1798 cells with 0.1 microM dexamethasone for 24 h results in an over 95% reduction of TFIC activity, but no significant differences in the amount of TFIC polypeptides in the final product purified from control and glucocorticoid-treated cells could be detected. Our data indicate that glucocorticoid regulation of transcription of rDNA is mediated via post-translational modulation of the activity of TFIC.
Mahajan
P B
PB
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.
Thompson
E A
EA
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004275:DNA, Ribosomal / Q000302:isolation & purification; D003907:Dexamethasone / Q000494:pharmacology*; D008228:Lymphoma, Non-Hodgkin / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D012318:RNA Polymerase I / Q000302:isolation & purification* / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
D004275:DNA, Ribosomal; D003907:Dexamethasone; D012318:RNA Polymerase I