Abstract
The ubiquitous transcription factor Oct-1 stimulates basal transcription from the mouse mammary tumor virus (MMTV) promoter by binding to octamer-related sequences present in the proviral long terminal repeat. The mechanism of transcriptional activation by Oct-1 was investigated using in vitro transcription assays with a HeLa cell nuclear extract depleted of endogenous Oct-1. Oct-1-mediated transcriptional activation could be reconstituted by addition of bacterially expressed recombinant Oct-1 protein. The stimulatory effect of Oct-1 was observed only when the protein was present during formation of transcription preinitiation complexes and not when added to fully assembled complexes. Furthermore, assembled MMTV preinitiation complexes were resistant to inhibition by a competitor oligonucleotide containing MMTV octamer-related elements that could eliminate Oct-1-mediated stimulation when present during the assembly process. The time course of transcription complex assembly revealed that Oct-1 increases the number of templates on which functional transcription complexes form. Finally, experiments designed to exploit the sensitivity of discrete steps in transcription complex assembly to the anionic detergent Sarkosyl demonstrated that Oct-1 must be present during formation of an early intermediate in the assembly process.
Highlights
Repression mediated by the distal negative regulatory elements (NREs) [12, 14]
These regulatory sequences modulate the transcriptional activity of the mammary tumor virus (MMTV) basal promoter, which contains sequences immediately 3Ј of the initiation site that are recognized by a nuclear protein termed initiation site binding protein, a TATA element, a binding site for nuclear factor 1 (NF-1), and two functional elements related to the octamer consensus (ATGCAAAT) between the TATA element and NF-1 binding site [15, 16]
Oct-1 Functions during Preinitiation Complex Assembly—To study the effects of Oct-1 on MMTV promoter activity, we have developed an Oct-1-responsive in vitro transcription system based on HeLa cell nuclear extract depleted of endogenous Oct-1 [28]
Summary
Repression mediated by the distal NRE [12, 14]. These regulatory sequences modulate the transcriptional activity of the MMTV basal promoter, which contains sequences immediately 3Ј of the initiation site that are recognized by a nuclear protein termed initiation site binding protein, a TATA element (centered near Ϫ30), a binding site for nuclear factor 1 (NF-1) (centered near Ϫ70), and two functional elements related to the octamer consensus (ATGCAAAT) between the TATA element and NF-1 binding site [15, 16]. Our transcription assays are generally performed in two stages; template DNA is incubated with nuclear extract proteins to allow assembly of transcription preinitiation complexes, and appropriate NTPs (including [␣-32P]CTP) are added to allow RNA synthesis.
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