ENHANCER 1 BINDING-FACTOR, A KU-RELATED PROTEIN, IS A POSITIVE REGULATOR OF RNA-POLYMERASE-I TRANSCRIPTION INITIATION

We have previously characterized a protein, enhancer 1 binding factor (E(1)BF), from rat tells that can modulate RNA polymerase I-directed t ranscription of the rat rRNA gene in vitro. E(1)BF, a heterodimeric DN A binding protein composed of 72-kDa and 85-kDa subunits, is related t o the human Ku autoantigen with respect to immunological and certain s tructural properties. To establish the direct role of E(1)BF in transc ription, we investigated the effect of anti gu antibodies on RNA polym erase I-directed transcription in rat and mouse cell extracts. These a ntibodies, one directed against the 70-kDa Ku subunit and the other ag ainst a peptide fragment of this subunit, dissociated the E(1)BF heter odimer into its two subunits. The DNA-protein complex formed in the pr esence of the antibodies contained only the 72-kDa submit. Preincubati on of the extracts with these antibodies resulted in an almost complet e inhibition of transcription. The reduced transcription was observed when either linear or circular template was used. The inhibitory effec t of the antibodies was greatest when added prior to preinitiation com plex formation and was minimized significantly when added after establ ishment of the initiation complex. The repression of rRNA gene transcr iption was overcome by the addition of purified E(1)BF. This study dem onstrates that E(1)BF, a Ku-related protein, is required for RNA polym erase I-directed transcription, the 72-kDa subunit is the major DNA bi nding polypeptide, the factor acts primarily in the formation of the p reinitiation complex, and heterodimerization of its two subunits is cr ucial for maintaining the functional integrity of the protein.