Complementary in vivo and in vitro analyses of the interactions between the cis-acting elements of the rat rDNA promoter.

Two transcription factors, rat UBF (rUBF) and rat SL-1 are required for the efficient transcription of the rat promoter in vitro. In vitro studies have established that two broadly defined cis-acting domains, the core promoter element and the upstream promoter element, cooperate to direct correct transcription by RNA polymerase I. The ability of UBF to bind to two linker-scanning mutants of the upstream promoter element, which did not respond to the addition of UBF in in vitro transcription assays, was assessed by DNase footprinting. UBF protected the same region of the promoter in the linker-scanning mutant in BSM 129/124 as it did in the wild-type, but did not yield a typical footprint over the promoter in the linker-scanning mutant BSM 106/101. Previously we reported that promoters with mutant core promoters elements, either the guanine at -16 or -7 substituted by an adenine, were inactive in vitro unless the assays were supplemented with UBF. Those results suggested that the binding of UBF upstream of the core was required for the promotion of transcription. The interactions between the core and upstream promoter elements were assessed by constructing double mutants of the promoter. In two constructs the conserved guanines at either -16 or -7 were altered in a deletion mutant (-86) that did not respond to UBF. In a third construct the guanine at -16 in BSM 129/124 was changed to an adenine. These bidomain mutant constructs did not respond to the addition of UBF in an in vitro transcription assay, confirming that the rescue of the core promoter mutants requires an intact and functional upstream promoter element.(ABSTRACT TRUNCATED AT 250 WORDS)