Start site selection by Sp1 in the TATA-less human Ha-ras promoter.
The promoter of the human Ha-ras gene is very GC-rich, has four Sp1-binding sites and no TATA element. Transcription initiates at multiple start sites spread over a 90-base pair region, with a major cluster between +1 and +11. We detected the binding of a HeLa nuclear protein to the region from +6 to +20, a region which overlaps the major cluster and includes nine start sites within its boundaries. When part of this region was mutated, the nine start sites were abolished, so the region may be an initiator. The regulatory elements of the promoter have no differential effect on start site usage. But Sp1 bound to GCIV located at -38 acts as a start site selector, increasing the activity of the start sites from +1 downstream but not the upstream sites at -31, -21, and -10. When spacers of increasing length were inserted 3' to GCIV, the major cluster dwindled, and the upstream sites became strong sites, first the one at -10 and then the one at -21. Thus start sites were strongest when put into an Sp1-selected locus 36 to 53 base pairs downstream from GCIV and weak when outside this locus. The start sites with these properties may be due to initiators.
Lu
J
J
AMC Cancer Research Center, Denver, Colorado 80214.
Lee
W
W
Jiang
C
C
Keller
E B
EB
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004275:DNA, Ribosomal / Q000235:genetics; D015870:Gene Expression; D011905:Genes, ras; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011544:Pseudogenes; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D012334:RNA, Neoplasm / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D016324:Sequence Tagged Sites; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D016385:TATA Box; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms
D004275:DNA, Ribosomal; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D016329:Sp1 Transcription Factor