ROLE OF THE TATA BOX IN TRANSCRIPTION OF THE MOUSE MAMMARY-TUMOR VIRUS LONG TERMINAL REPEAT

An in vitro transcription system from mammary cells was established to study transcription of the long terminal repeat (LTR) of the mouse ma mmary tumor virus (MMTV). Experiments with progressive 5'-deletion con structs of the MMTV LTR revealed that a 19-base pair (bp) region from -41 to -23 bp, encompassing the TATA box and flanking DNA sequence, wa s as transcriptionally active as larger promoter constructs, both in n uclear extracts from human mammary cell lines (T47D and MCF7) and a no nmammary cell line (HeLa). The cell-free system was capable of support ing transcriptional induction by factors binding upstream of the TATA box, however, since purified glucocorticoid receptor-induced transcrip tion in larger promoter constructs encompassing the MMTV hormone-respo nsive elements. Transcription from two other promoters, the adenovirus major late promoter and the human immunodeficiency virus LTR, also re vealed a significant transcriptional contribution of upstream elements . The 19-bp TATA region from the MMTV LTR was shown to have considerab ly more activity in this transcription system than comparable TATA reg ions from other promoters. Sequences critical to the MMTV TATA region were evaluated by single base pair mutagenesis and found to comprise a consensus TATA box sequence, TATAAAA, as well as a single A just upst ream of the TATAAAA sequence. Thus, the high level of basal transcript ion observed with the TATA region from MMTV is due to a perfect consen sus TATA box sequence and a single base immediately 5' adjacent. It is likely that the high basal rate of transcription observed with this T ATA box region on histone-free templates represents an inappropriate l ever of basal expression and that a complete evaluation of transactiva tion mechanisms in this system will require the recapitulation in vitr o of the chromatin-mediated repressive state that exists in vivo.