| An in vitro transcription system for the mouse mammary tumor virus (MMTV) promoter was developed using nuclear extracts prepared from mammalian tissue culture cell lines (HeLa and Ltk{dollar}sp{lcub}-{rcub}{dollar}) and "MMTV-Tfree cassette" template constructs. Transcriptional repression of MMTV promoter activity of approximately 2- to 3-fold, which was dependent on the presence of the distal negative regulatory element (dNRE) in the MMTV LTR, was observed with this system in numerous experiments with independent preparations of nuclear extracts and plasmid templates. Several kinetic experiments were performed using this in vitro transcription system, in which the time courses of transcription complex assembly and transcript elongation were determined. These experiments indicated that the dNRE causes transcription repression in vitro by affecting the assembly of transcription complexes. A protein binding to one previously identified mutation-sensitive domain of the dNRE was identified in nuclear extracts of both HeLa and Ltk{dollar}sp{lcub}-{rcub}{dollar} cell lines. In phosphocellulose chromatography, this protein was eluted with 0.5 M KCl in the case of HeLa nuclear extract and 1.0 M KCl in the case of Ltk{dollar}sp{lcub}-{rcub}{dollar} nuclear extract. Furthermore, the sequence recognized by this protein was clearly defined within the dNRE between {dollar}-{dollar}434 and {dollar}-{dollar}418 relative to the transcription initiation site. When inserted in front of MMTV basal promoter, this sequence repressed in vitro transcription, but not as efficiently as the entire 91 bp dNRE. As a first step toward characterization of the protein binding to this sequence, the apparent molecular weight of the protein was estimated as about 100 kDa in gel filtration chromatography, and as about 125 kDa in Southwestern analysis. A model for dNRE function was developed based on binding of trans-acting factors to the dNRE and inhibitory protein-protein interactions with a transcription factor(s) during preinitiation complex formation at the basal promoter, thereby decreasing the number of functional preinitiation complexes. |
