FUNCTIONAL-CHARACTERIZATION OF THE CIS-REGULATORY ELEMENTS OF THE RATRIBOPHORIN-I GENE

The ribophorin I gene encodes a rough endoplasmic reticulum (RER) spec ific membrane protein which is a subunit of the oligosaccharyltransfer ase. To establish the functional activity of its promoter region we ha ve performed transient gene transcription experiments employing plasmi d constructs that contain 5' flanking regions of the ribophorin I gene cloned upstream of the CAT reporter gene. Among the restriction fragm ents obtained from the 1.3-kilobase 5' flanking region, a proximal fra gment (-42 to +24) containing two CC-rich elements was required for ba sic promoter activity, while a fragment (-364 to +24) encoding an addi tional GC-box and an octamer like motif at -233 conferred the maximal promoter activity. In order to investigate the functionality of an oct omer-like sequence co-transfection experiments were performed with Oct -2 cDNA and the CAT reporter gene containing the ribophorin I fragment (-364 to +24). A 3-4-fold increase in the transcriptional activity wa s observed with this construct. In addition, gel shift experiments sho wed Oct-2 binding to this construct. These results indicate that Oct-2 is most likely involved in the regulation of the ribophorin I gene tr anscription. We suggest that the CC-rich elements are necessary for co nstitutive ribophorin I expression while octamer motif binding protein s function synergistically with the CC-rich element binding proteins t o increase the expression of the ribophorin I gene during the prolifer ation of RER.