Abstract
Transcription by RNA polymerase 11 of the promoter within the proviral DNA of the mouse mammary tumor virus long terminal repeat (MNM LTR) requires numerous short, DNA sequence elements that can be bound by sequence-specific protein factors. One such factor binds a promoter element just downstream of the transcription initiation site of MMTV - initiation site binding protein (ISBP). This site is required for optimal basal and hormoneinduced transcription from the MMTV LTR promoter. The goal of this research was to determine what roles ISBP and the initiator-like element that it binds play in transcription from the MNM LTR promoter. ISBP was isolated from bovine liver nuclear extract and an ISBP polypeptide of 21.5 kD/ 23.5 kD was identified. A preliminary attempt to isolate an ISBP CDNA clone based upon DNA-binding was attempted. To determine if ISBP was an already characterized and/ or cloned initiator or initiator-like binding protein, the ability of ISBP to bind additional promoters with initiator or initiator-like sequences was evaluated by DNase I footprinting. ISBP specifically recognized the initiator sites on the terminal deoxynucleotidyltransferase, SV40 major late, and adenovirus 2 ElB promoters, but not on the adenovirus major late, dihydrofolate reductase, and adeno-associated virus type 2 P5 promoters. Based upon these results and the small size of ISBP, ISBP was not a previously characterized initiator or initiatorlike binding protein. To assess whether the ISBP site of M@ had initiator activity, spacing mutations between the TATA box and the initiator/ ISBP site were tested in vivo. The ISBP site had some properties of classic initiators with some properties unique to MMTV. To develop an in vitro assay for ISBP activity, an in vitro transcription system derived from fractionated bovine liver nuclear extract was characterized. The system proved responsive to both TATA-binding protein (TBP) and TFIIB. In this highly fractionated extract, the response to addition of ISBP was not detected. However, ISBP activity was observed using less-fractionated extract.
Fee, Brian Edward (1995). Initiation site binding protein and the initiator-like promoter element of mouse mammary tumor virus. Texas A&M University. Texas A&M University. Libraries. Available electronically from
https : / /hdl .handle .net /1969 .1 /DISSERTATIONS -1561503.