TRANSCRIPTIONAL ACTIVITY OF THE HOMOPURINE - HOMOPYRIMIDINE REPEAT OFTHE C-KI-RAS PROMOTER IS INDEPENDENT OF ITS H-FORMING POTENTIAL

The mouse c-Ki-ras protooncogene promoter contains an unusual DNA elem ent consisting of a 27 bp-long homopurine - homopyrimidine mirror repe at (H-motif) adjacent to a d(C-G)(5) repeat. We have previously shown that in vitro these repeats may adopt H and Z conformations, respectiv ely, causing nuclease and chemical hypersensitivity. Here we have stud ied the functional role of these DNA stretches using fine deletion ana lysis of the promoter and a transient transcription assay in vivo. We found that while the H-motif is responsible for approximately half of the promoter activity in both mouse and human cell lines, the Z-formin g sequence exhibits little, if any, such activity. Mutational changes introduced within the homopurine - homopyrimidine stretch showed that its sequence integrity, rather than its H-forming potential, is respon sible for its effect on transcription. Electrophoretic mobility shift assays revealed that the putative H-motif tightly binds several nuclea r proteins, one of which is likely to be transcription factor Sp1, as determined by competition experiments. Southwestern hybridization stud ies detected two major proteins specifically binding to the H-motif: a 97 kD protein which presumably corresponds to Sp1 and another protein of 60 kD in human and 64 kD in mouse cells. We conclude that the homo purine - homopyrimidine stretch is required for full transcriptional a ctivity of the c-Ki-ras promoter and at least two distinct factors, Sp l and an unidentified protein, potentially contribute to the positive effect on transcription.