IDENTIFICATION OF A MINIMAL SET OF PROTEINS THAT IS SUFFICIENT FOR ACCURATE INITIATION OF TRANSCRIPTION BY RNA POLYMERASE-II

In eukaryotes, initiation of mRNA synthesis is a multistep process tha t is carried out by RNA polymerase II and auxiliary factors that are c ommonly referred to as basal or general factors. In this study accurat e initiation of transcription was reconstituted with purified, Escheri chia coli-synthesized TFIIB, TBP (the TATA box-binding polypeptide of the TFIID complex), and the 30-kD subunit of TFIIF (also known as RAP3 0), along with purified, native RNA polymerase II from Drosophila embr yos, calf thymus, or HeLa cells. This minimal set of factors was able to transcribe a subset of the promoters tested. The addition of both s ubunits of TFIIE and the 74-kD subunit of TFIIF increased the efficien cy of transcription by a factor of 2 to 4. In contrast, the inclusion of a crude TFIID fraction from Drosophila embryos in place of recombin ant TBP resulted in a strong dependence on TFIIE. By gel mobility-shif t analysis, TFIIB, TBP, RAP30, and polymerase were able to assemble in to DB and DBPolF30 complexes with transcriptionally competent (wild ty pe or initiator mutant), but not with transcriptionally inactive (TATA and TATA/initiator mutant), versions of the Drosophila Adh promoter. Thus, it appears that RNA polymerase II is able to initiate transcript ion subsequent to assembly of the DBPolF30 complex, which is a minitra nscription complex that represents the central core of the RNA polymer ase II transcriptional machinery.