Purification of Xenopus transcription factor IIIA and 5 S RNA from 7 S ribonucleoprotein particle by ammonium sulfate precipitation.
A simple and efficient method for purifying Xenopus transcription factor IIIA from the 7 S particle has been developed by taking advantage of the differential solubilities of the protein factor and 5 S RNA in ammonium sulfate solution. Conditions under which ammonium sulfate dissociates the 7 S particle and selectively precipitates factor IIIA while the 5 S RNA moiety remains in the supernatant were found. The method simultaneously purifies, in a nondestructive manner, both factor IIIA and 5 S RNA in high yield. Purification proceeds through several ammonium sulfate precipitations of the 7 S particle. Factor IIIA obtained by this method contains no detectable RNA and is highly active as judged by DNase I footprinting and in vitro transcription of the 5 S RNA gene, as well as reconstitution with 5 S RNA to form the 7 S particle. The molar extinction coefficients of factor IIIA at 205 and 280 nm were determined from the ultraviolet absorption spectra measured with the purified protein.
D000645:Ammonium Sulfate; D000818:Animals; D011232:Chemical Precipitation; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D066298:In Vitro Techniques; D008970:Molecular Weight; D009865:Oocytes / Q000032:analysis; D012313:RNA / Q000302:isolation & purification*; D012261:Ribonucleoproteins / Q000032:analysis*; D013056:Spectrophotometry, Ultraviolet; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000302:isolation & purification* / Q000502:physiology; D014158:Transcription, Genetic; D014982:Xenopus laevis
D012261:Ribonucleoproteins; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors; D012313:RNA; D000645:Ammonium Sulfate