THE NOVEL PURIFICATION OF FUNCTIONAL RNA POLYMERASE-III VAI TRANSCRIPTION PREINITIATION COMPLEXES

A novel purification scheme employing high-performance liquid chromatography (HPLC) gel filtration chromatography on a 4000 A methacrylate-based polymer and preparative gel mobility shift fractionation on 2% agarose gels with continuous elution allowed for a 10(5) fold enrichment in RNA polymerase III transcription preinitiation complexes from NTP-depleted HeLa whole cell extracts. Purified preinitiation complexes from the TATA-less Class III (Internal Control Region) adenovirus VA(I) gene contain the TATA-Box binding protein (TBP) by the criterion of comigration on sodium dodecyl sulfate (SDS) gels with the 43 kd TBP present in a purified B-TFIID fraction, and by Western blot analysis. Purified VAI preinitiation complex preparations lacking RNA polymerase III consistently contain a number of additional polypeptides with apparent molecular weights of 17, 20, 29,32, 39, 55, 105, 120, and 140 kDa which appear to be specific components, since they are not detected in the corresponding fractions isolated using a VAI gene B-block mutant promoter.