Functional characterization of the cis-regulatory elements of the rat ribophorin I gene.
The ribophorin I gene encodes a rough endoplasmic reticulum (RER) specific membrane protein which is a subunit of the oligosaccharyltransferase. To establish the functional activity of its promoter region we have performed transient gene transcription experiments employing plasmid constructs that contain 5' flanking regions of the ribophorin I gene cloned upstream of the CAT reporter gene. Among the restriction fragments obtained from the 1.3-kilobase 5' flanking region, a proximal fragment (-42 to +24) containing two GC-rich elements was required for basic promoter activity, while a fragment (-364 to +24) encoding an additional GC-box and an octamer like motif at -233 conferred the maximal promoter activity. In order to investigate the functionality of an octomer-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 was observed with this construct. In addition, gel shift experiments showed Oct-2 binding to this construct. These results indicate that Oct-2 is most likely involved in the regulation of the ribophorin I gene transcription. We suggest that the GC-rich elements are necessary for constitutive ribophorin I expression while octamer motif binding proteins function synergistically with the GC-rich element binding proteins to increase the expression of the ribophorin I gene during the proliferation of RER.
Rajasekaran
A K
AK
Department of Cell Biology, New York University School of Medicine, New York 10016.
Zhou
Z
Z
Prakash
K
K
Das
G
G
Kreibich
G
G
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum; D005347:Fibroblasts; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D050813:Octamer Transcription Factor-2; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014157:Transcription Factors; D014158:Transcription, Genetic / Q000235:genetics*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D050813:Octamer Transcription Factor-2; C058435:POU2F2 protein, human; C489205:Pou2f2 protein, rat; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C034241:ribophorin; D004247:DNA