Transcription of the human ferredoxin gene through a single promoter which contains the 3',5'-cyclic adenosine monophosphate-responsive sequence and Sp 1-binding site.
We have investigated the functional elements involved in cAMP-stimulated transcription of the human ferredoxin gene. Unlike the bovine gene, the human gene lacked a second upstream RNA initiation site as demonstrated by sequence analysis of the exon boundary, lack of upstream RNA, and analysis of the promoter. The presence of a single promoter was determined by testing the ability of various gene segments to drive the expression of the chloramphenicol acetyltransferase gene after transfection into a mouse adrenal cell line Y1. Full promoter activity was conferred by a DNA fragment spanning -209 to +55, although the -94 to +55 fragment already provided some promoter activity. Transcription from the -94 to +55 segment was stimulated by 2-fold when 8-bromo-cAMP was added to the cell. Footprinting analyses showed two GC boxes at -50 to -70 and -87 to -108 were protected by proteins from both Y1 and HeLa cells. Competition experiments showed that a protein with a recognition sequence indistinguishable from Sp1 bound to these sites. When connected to a heterologous TATA box, the sequence at -76 to -42, which contained the proximal GC box, was able to confer a high level of basal transcription and cAMP stimulation. This sequence does not show sequence homology with the known cAMP-responsive element. Mutations or deletion of the Sp 1-binding site showed diminished basal transcription and defined the cAMP responsive sequence to be from -76 to -62. Therefore the cAMP-responsive sequence of the human ferredoxin gene was located at -76 to -62, which was adjacent to the Sp 1-binding site.
Chang
C Y
CY
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China.
Huang
C
C
Guo
I C
IC
Tsai
H M
HM
Wu
D A
DA
Chung
B C
BC
D000595:Amino Acid Sequence; D001483:Base Sequence; D000242:Cyclic AMP / Q000502:physiology; D004268:DNA-Binding Proteins / Q000378:metabolism; D005288:Ferredoxins / Q000235:genetics*; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D005288:Ferredoxins; D016329:Sp1 Transcription Factor; D000242:Cyclic AMP