MULTIPLE REGULATORY ELEMENTS ENSURE ACCURATE TRANSCRIPTION OF A HUMANRIBOSOMAL-PROTEIN GENE

Previously we have shown that expression of a cloned human ribosomal p rotein gene, RPS14, depends upon regulatory sites located within the g ene's proximal upstream DNA plus its first intron. In order to identif y cis-active sequence motifs within the RPS 14 promoter-enhancer compl ex, we transiently expressed a set of informative deletion clones in c ultured Chinese hamster ovary cells. These experiments revealed three DNA sequence motifs that surround the S14 mRNA initiation site and are necessary for accurate transcription. Electrophoretic mobility shift, DNase I footprint, and methylation interference assays resolved two n uclear proteins, NFalpha-1 and NFbeta-1, which bind specifically to th ese regulatory motifs. NF-alpha1 recognizes a pair of 6-bp target moti fs (5'-TTCCGG-3') that flank the 5' end of RPS14 exon I; and NF-beta1 binds to a 10-bp target sequence (5'-CCGTGGGAAC-3') within the gene's first intron. Site-directed deletion mutations within the NF-alpha1 an d -beta1 binding sites markedly inhibit S14 mRNA transcription.