INITIATION OF TRANSCRIPTION BY RNA-POLYMERASE-II IS LIMITED BY MELTING OF THE PROMOTER DNA IN THE REGION IMMEDIATELY UPSTREAM OF THE INITIATION SITE

To further elucidate the mechanism of transcriptional initiation, we u sed synthetic oligonucleotides to prepare templates containing heterod uplex regions of varying size and location along the DNA of the adenov irus major late promoter. Unlike closed, linear DNA, or DNA with a dow nstream mismatch, DNA with a mismatch upstream of the initiation site only required the general factors TATA box-binding protein and transcr iption factor (TF) IIB to direct specific and accurate initiation in v itro by calf thymus RNA polymerase II. In the presence of TFIIF, initi ation was possible on closed, linear DNA, but an upstream mismatch reg ion still stimulated transcriptional initiation by more than 100-fold, leading to production of similar to 0.5 transcript/template in the ab sence of TFIIE, TFIIH, or ATP. The presence of a DNA mismatch was most effective in the -9 to -1 region; furthermore, stimulation by a misma tch did not require that the initiation site be included in the hetero duplex region. Efficient initiation at the immunoglobulin heavy chain promoter in the presence of TATA box-binding protein and TFIIB was als o achieved when a mismatch region was introduced from -9 to +3. Our re sults suggest that initiation by RNA polymerase II in the absence of t ranscriptional activation is limited by melting of the promoter DNA up stream of the initiation site.