Abstract
Transcription of the proviral genes of Mouse Mammary Tumor Virus (MMTV) is subject to induction by several classes of hormone-activated steroid receptor proteins, as well as to repression mediated by sequences that inhibit the inherent, basal activity of the proviral promoter in the absence of a hormonal stimulus. Elucidation of the molecular mechanisms by which activity of the proviral promoter is modulated will first require a detailed understanding of promoter function in the absence of regulatory signals. An in vitro transcription system for analysis of MMTV promoter activity has been developed based on a supercoiled, T-free cassette template in which proteins necessary for transcription are supplied by a nuclear extract supplemented, in some cases, with purified transcription factors expressed in bacteria from cloned cDNAs. With this system it is demonstrated that transcription factors (TF) IID and IIB limit the rate of assembly of complexes competent to rapidly initiate transcription. Mutations in MMTV promoter elements recognized by nuclear factor 1, octamer binding protein 1, or initiation site binding protein result in a decrease in the number of promoter-containing templates on which functional transcription complexes can form in vitro. However, this is not accompanied by a decrease in the TFIID- or TFIIB-limited rate of transcription complex assembly, indicating that NF-1, Oct-1 and ISBP do not affect the association of these basal factors with the MMTV promoter. Mutation of the binding site of ISBP leads to an increased rate of RNA synthesis relative to wild-type templates. An equilibrium model is proposed to aid interpretation of these studies, and using this model a potential role for ISBP in open complex stabilization is defined.
Bral, Christopher Michael (1994). Preinitiation complex formation on the mouse mammary tumor virus promoter in vitro. Texas A&M University. Texas A&M University. Libraries. Available electronically from
https : / /hdl .handle .net /1969 .1 /DISSERTATIONS -1556365.