PHOSPHORYLATION OF HUMAN GENERAL TRANSCRIPTION FACTORS TATA-BINDING PROTEIN AND TRANSCRIPTION FACTOR IIB BY DNA-DEPENDENT PROTEIN-KINASE - SYNERGISTIC STIMULATION OF RNA-POLYMERASE-II BASAL TRANSCRIPTION IN-VITRO

DNA-dependent protein kinase (DNA-PK) has been known to catalyze phosp horylation of a number of regulator factors involved in DNA replicatio n and transcription such as simian virus 40 T antigen, p53, c-Myc, Sp1 , and RNA polymerase II (Pol II). We examined the possibility that DNA -PK phosphorylates the general transcription factors TATA-binding prot ein (TBP) and transcription factor (TF) IIB. which play key roles in t he formation of transcription initiation complex with Pol II. By using a highly purified preparation of DNA-PK from Raji cells, both TBP and TFIIB were shown to be phosphorylated in vitro by DNA-PK. We then inv estigated the effect of the phosphorylation of these factors on Pol II basal transcription, Stepwise analysis of preinitiation complex forma tion by electrophoretic mobility shift assay revealed that the phospho rylation of TBP and TFIIB by DNA-PK did not affect the formation of pr omoter (P)-TBP and P-TBP-TFIIB complexes but synergistically stimulate d the formation of P-TBP-TFIIB-TFIIF-Pol II complex. Similarly, combin ation of the phosphorylated TBP and TFIIB, synergistically stimulated Pol II basal transcription from adenovirus major late promoter. These observations suggest that DNA-PK could positively regulate the pol II basal transcription by phosphorylating TBP and TFIIB.