PHYSICAL SEPARATION OF 2 DIFFERENT FORMS OF HUMAN TFIIIB ACTIVE IN THE TRANSCRIPTION OF THE U6 OR THE VAI GENE IN-VITRO

Human transcription factor hTFIIIB is necessary to initiate transcript ion correctly from all RNA polymerase III (pol III) genes which are go verned by structurally different promoters, and it is unclear whether hTFIIIB complexes, required for intragenic or 5'-located pol III promo ters, are composed of unique or different components, We show here tha t two different forms of hTFIILB can be separated physically by ion ex change chromatography. hTFIIIB-alpha shows strong preference for trans cription of the U6 over the VAI gene and does not contain TATA binding protein (TBP), After SDS-PAGE and renaturation of proteins, the trans criptional activity of hTFIIIB-alpha can be reconstituted by fractions corresponding to a mean M(r) of 25, 60 and 90 kDa, Upon gradient cent rifugation or gel filtration, the activity of hTFIILB-alpha is associa ted with an M(r) of 60 +/- 10 kDa, indicating that the components of t he complex tend to dissociate. In contrast, hTFIIIB-beta is predominan tly active on intragenic pol LII promoters. It reveals an M(r) of 300 +/- 30 kDa upon gel filtration and, besides TBP, it contains several a ssociated factors (TAFs). Two of these proteins reveal an M(r) of 60 k Da and 90 kDa, and it is conceivable that they are related to polypept ides of similar mass functionally identified in hTFIIIB-alpha. These p roteins are probably required for the recruitment of pol III to the in itiation site at 5'-located and intragenic promoters.