FIG. 1.

Immobilized human rRNA gene promoter fragments containing the UCE and core promoter elements support in vitro transcription. The promoter fragments generated by PCR (Fr2 to Fr4) are schematically outlined. PCR primer binding sites, relative to the transcription start site at +1, and the UCE (−156 to −107) and core region (−45 to +18) in the ribosomal promoter are indicated. prHu3 is a pBR322-derived supercoiled plasmid DNA containing the human ribosomal promoter sequence from −515 to +1548. In vitro transcription assays contained 1 μl of HeLa cell nuclear extract (NE), and a 1.25 pM concentration of the appropriate template. 5′-end-biotinylated DNA was immobilized (5′ imm.) onto 2.5 μl of streptavidin-coated paramagnetic beads (Dynabeads M280; Dynal). Transcripts synthesized in vitro were analyzed in S1 nuclease protection assays with a radiolabeled oligonucleotide overlapping the transcription start site (see Materials and Methods).