Regulatory elements mediating transcription of the human Ha-ras gene

In order to identify transcriptional regulatory elements controlling the expression of the human Ha-ras gene and to quantitatively assess the role of each element, we made mutations of the transcriptional regulatory region, including 5' and internal deletions, linker scanning and replacement mutations, and combinations of these mutations all fused to the bacterial chloramphenicol acetyltransferase gene. The promoter activity of each of these mutants was determined by measuring the transient expression of chloramphenicol acetyltransferase activity after transfection into human epithelial HeLa cells. We found that the most important regulatory region consists of two closely linked but functionally independent elements, the non-consensus GC-II element, CGGGCGGGC, centered at position -153 from the major transcription start site cluster and a new element, CCGGAA, centered at position -161 directly upstream from GC-II. In addition, there are two functional regulatory elements which make minor contributions to the full promoter activity; a double CCAAT NF-I binding site at position -88 and an unidentified upstream element between positions -199 and -252. Aside from GC-II, the GC boxes, of which there are a total of six between positions -185 and +85, make little or no contribution to Ha-ras promoter activity when individual mutations are tested in growing HeLa cells. The three potential AP2 sites and a weak single NF-I binding site make no contribution. The basal promoter region extending to position -75 from the major start site cluster has no independent activity in this TATA-less gene.