Association of nucleosome-free regions and basal transcription factors with in vivo-assembled chromatin templates active in vitro

Abstract

Using SV40 minichromosomes assembled in vivo, we have studied the relationship between a nucleosome-free promoter-region and initiation of transcription by RNA polymerase II on chromatin templates in vitro. Our data suggest that accessibility of DNA to transcription factors, programmed into the structure of the chromatin, is crucial for initiation of transcription. First, minichromosomes competent to be transcribed in vitro contained nucleosome-free promoter regions. Second, tsC219 minichromosomes, most of which contain the nucleosome-free promoter region, supported transcription more efficiently both in vivo and in vitro than wild-type minichromosomes, in which only a subset contain the nucleosome-free region. We have also identified basal transcription factors associated with the in vivo-assembled chromatin templates. A striking correlation was observed between minichromosomes associated with in vivo initiated RNA polymerases and those associated with the basal transcription factors TFIID and TFIIE/F, and to a lesser extent, TFIIB. Of these associated factors, only TFIID was poised for ready assembly into preinitiation complexes and therefore for subsequent initiation of transcription. However, an active chromatin template could also be maintained in the absence of the binding of TFIID. Finally, our data are consistent with the presence of TFIIF in elongating ternary complexes on the chromatin templates.