Functional characterization of transcriptional regulatory elements in the upstream region and intron 1 of the human S6 ribosomal protein gene

Expression of housekeeping genes involves regulation at comparable levels i n a wide spectrum of cells. To define the cis-regulatory elements in the hu man S6 ribosomal protein (rpS6) gene, we made a series of deletions of the upstream nontranscribed region, including or excluding exon 1 or intron 1 s equences. The mutated rpS6 gene regulatory regions were fused to the chlora mphenicol acetyltransferase reporter gene and transfected into HeLa and COS -1 cells. The results have identified three parts of the rpS6 gene that are required for efficient and specific transcription. The core promoter inclu des only a 40 bp region upstream of the transcription start site and initia tion region. Both upstream and intronic elements enhance transcription from the core promoter. Furthermore, mutation of the splice donor site of intro n I almost completely abolished the enhancing activity of the intronic tran scriptional modulator. We used gel retardation assays to identify sequence- specific binding sites in the upstream region and in the proximal half of i ntron I. Both common and different nuclear factors that bind the rpS6 gene promoter were identified in extracts from HeLa and COS-1 cells, suggesting that different transcription factors may bind specifically to the same bind ing region and might be interchangeable in their function to ensure high-le vel expression of housekeeping genes independently of the cell type.