Transcriptional activity of the homopurine-homopyrimidine repeat of the c-Ki-ras promoter is independent of its H-forming potential.
The mouse c-Ki-ras protooncogene promoter contains an unusual DNA element consisting of a 27 bp-long homopurine-homopyrimidine mirror repeat (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, respectively, causing nuclease and chemical hypersensitivity. Here we have studied the functional role of these DNA stretches using fine deletion analysis 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-forming 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 responsible for its effect on transcription. Electrophoretic mobility shift assays revealed that the putative H-motif tightly binds several nuclear proteins, one of which is likely to be transcription factor Sp1, as determined by competition experiments. Southwestern hybridization studies 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 homopurine-homopyrimidine stretch is required for full transcriptional activity of the c-Ki-ras promoter and at least two distinct factors, Sp1 and an unidentified protein, potentially contribute to the positive effect on transcription.
Raghu
G
G
Department of Genetics, University of Illinois at Chicago 60612.
Tevosian
S
S
Anant
S
S
Subramanian
K N
KN
George
D L
DL
Mirkin
S M
SM
D001483:Base Sequence; D001665:Binding Sites; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism; D011905:Genes, ras; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D011687:Purines / Q000737:chemistry*; D011743:Pyrimidines / Q000737:chemistry*; D012091:Repetitive Sequences, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D004278:DNA, Superhelical; D009687:Nuclear Proteins; D011687:Purines; D011743:Pyrimidines; D016329:Sp1 Transcription Factor