Transcription factor requirement for multiple rounds of initiation by human RNA polymerase II.

We have investigated conditions that allow multiple rounds of transcription initiation from the adenovirus major late promoter in an in vitro system derived from HeLa cell nuclear extracts. Templates containing guanine-free cassettes provided a direct assay for discriminating between reinitiated transcripts and transcripts generated by a first-round of transcription initiations. When reactions were reconstituted with the previously characterized class II transcription factors (TFIIA, TFIIB, TFIID, TFIIE/F), transcription by human RNA polymerase II from the adenovirus major late promoter was essentially restricted to a single round of initiations. Reinitiations at previously transcribed major late templates required an additional activity, designated reinitiation transcription factor (RTF). The RTF activity could be separated from the required transcription initiation factors. Semipurified human RTF also promoted transcription reinitiations at minimal promoters derived from the human c-myc, histone H4, and heat shock 70-kDa protein genes, indicating that the same reinitiation factor may be utilized by many, if not all, genes. The possible role of RTF in regulating the transcription rate of various class II genes is discussed.