Start site selection at the TATA-less carbamoyl-phosphate synthase (glutamine-hydrolyzing)/aspartate carbamoyltransferase/dihydroorotase promoter.
Transcription of the carbamoyl-phosphate synthase (glutamine-hydrolyzing)/aspartate carbamoyltransferase/dihydroorotase (CAD) gene from the Syrian hamster, Mesocricetus auratus, starts at a single major site. We characterized the cis-acting elements that position RNA polymerase II at the correct start site in the CAD promoter. Sequence alignment showed that the CAD promoter lacks a TATA box, but contains a consensus initiator. Mutational analysis of the CAD promoter demonstrated that the sequences between -81 and +26 were sufficient for accurate and efficient transcription in vitro and in vivo; binding sites for the transcription factor Sp1 around -70 and -49 were necessary for transcriptional activity. The binding site at -49 directed initiation about 50 base pairs downstream. A ubiquitous activator protein, Honk, bound to the CAD promoter between -30 and -12, but did not participate in start site selection. The sequences around +1, which contain the consensus initiator, contributed to promoter activity; however, the presence of a consensus initiator in this region was neither necessary nor sufficient for transcription. We concluded from these results that the Sp1 binding site at -49 substituted for the missing TATA box and played a major role in start site selection at the CAD promoter.
Kollmar
R
R
Program in Cell and Molecular Biology, University of Wisconsin, Madison 53706.
Sukow
K A
KA
Sponagle
S K
SK
Farnham
P J
PJ
D016475:3T3 Cells; D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000096:biosynthesis / Q000235:genetics*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D016384:Consensus Sequence; D006224:Cricetinae; D004247:DNA / Q000378:metabolism; D004080:Dihydroorotase / Q000096:biosynthesis / Q000235:genetics*; D008099:Liver / Q000378:metabolism; D008297:Male; D008647:Mesocricetus / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000235:genetics*; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013601:T-Lymphocytes / Q000378:metabolism; D016385:TATA Box; D014162:Transfection
C033626:CAD trifunctional enzyme; D009097:Multienzyme Complexes; D009838:Oligodeoxyribonucleotides; D004247:DNA; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)