Multiple regulatory elements ensure accurate transcription of a human ribosomal protein gene

Previously we have shown that expression of a cloned human ribosomal protein gene, RPS14, depends upon regulatory sites located within the gene's proximal upstream DNA plus its first intron. In order to identify cis-active sequence motifs within the RPS14 promoter-enhancer complex, we transiently expressed a set of informative deletion clones in cultured 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 nuclear proteins, NF alpha-1 and NF beta-1, which bind specifically to these regulatory motifs. NF-alpha 1 recognizes a pair of 6-bp target motifs (5'-TTCCGG-3') that flank the 5' end of RPS14 exon I; and NF-beta 1 binds to a 10-bp target sequence (5'-CCGTGGGAAC-3') within the gene's first intron. Site-directed deletion mutations within the NF-alpha 1 and -beta 1 binding sites markedly inhibit S14 mRNA transcription.