START SITE SELECTION AT THE TATA-LESS CARBAMOYL-PHOSPHATE SYNTHASE (GLUTAMINE-HYDROLYZING) ASPARTATE CARBAMOYLTRANSFERASE DIHYDROOROTASE PROMOTER

Transcription of the carbamoyl-phosphate synthase (glutamine-hydrolyzi ng)/aspartate carbamoyltransferase/dihydroorotase (CAD) gene from the Syrian hamster, Mesocricetus auratus, starts at a single major site. W e characterized the cis-acting elements that position RNA polymerase I I at the correct start site in the CAD promoter. Sequence alignment sh owed that the CAD promoter lacks a TATA box, but contains a consensus initiator. Mutational analysis of the CAD promoter demonstrated that t he sequences between -81 and +26 were sufficient for accurate and effi cient transcription in vitro and in vivo; binding sites for the transc ription factor Spl around -70 and -49 were necessary for transcription al 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 sit e selection. The sequences around +1, which contain the consensus init iator, contributed to promoter activity; however, the presence of a co nsensus initiator in this region was neither necessary nor sufficient for transcription. We concluded from these results that the Spl bindin g site at -49 substituted for the missing TATA box and played a major role in start site selection at the CAD promoter.