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Resolution of transcription factors from a transcriptionally active whole-cell extract from yeast: purification of TFIIB, TBP, and RNA polymerase IIa.

We describe techniques for production and chromatographic fractionation of a transcriptionally active whole-cell extract from Saccharomyces cerevisiae. The procedure is suitable for large-scale isolation of the factors involved in mRNA synthesis. Both yeast transcription factor IIB and TATA-binding protein were purified from the extract as single species using an immunoblot assay. In addition, the three previously described isoforms of yeast RNA polymerase II were resolved and form IIa, the intact, unphosphorylated isoform proposed to be involved in initiation, was purified to apparent homogeneity.