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

A novel purification scheme employing high-performance liquid chromato graphy (HPLC) gel filtration chromatography on a 4000 A methacrylate-b ased polymer and preparative gel mobility shift fractionation on 2% ag arose 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 fr om the TATA-less Class III (Internal Control Region) adenovirus VA(I) gene contain the TATA-Box binding protein (TBP) by the criterion of co migration on sodium dodecyl sulfate (SDS) gels with the 43 kd TBP pres ent in a purified B-TFIID fraction, and by Western blot analysis. Puri fied VAI preinitiation complex preparations lacking RNA polymerase III consistently contain a number of additional polypeptides with apparen t molecular weights of 17, 20, 29,32, 39, 55, 105, 120, and 140 kDa wh ich appear to be specific components, since they are not detected in t he corresponding fractions isolated using a VAI gene B-block mutant pr omoter.