Purification of Xenopus transcription factor IIIA and 5 S RNA from 7 S ribonucleoprotein particle by ammonium sulfate precipitation

Zhigang Shang, William T. Windsor, You Di Liao, Cheng-Wen Wu Lee*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)156-163
Number of pages8
JournalAnalytical Biochemistry
Volume168
Issue number1
DOIs
StatePublished - 1 Jan 1988

Keywords

  • 7 S particle
  • ammonium sulfate precipitation
  • transcription factor IIIA

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