Leucine zipper domain of 52 kDa SS-A/Ro promotes protein dimer formation and inhibits in vitro transcription activity.
Two forms of the human 52 kDa SS-A/Ro protein autoantigen, 52alpha and 52beta, are products of alternative mRNA splicing. The 52alpha form is ubiquitously expressed whereas 52beta, lacking the central leucine zipper domain, has been detected at higher levels than 52alpha during certain stages of fetal development. Because 52alpha has sequence similarity with macromolecules associated with transcriptional regulation and the two forms differ only in that 52beta does not contain the leucine zipper, their roles in protein dimer formation and in transcriptional activity were examined. Employing the yeast two-hybrid system, 52alpha was shown to interact with itself but not 52beta. The homodimerization of 52alpha was independently confirmed in gel filtration chromatography using in vitro cDNA template derived translation products and in HL-60 cell extracts; two peaks were observed corresponding to dimer and monomer of 52alpha, while in vitro the translation product of 52beta exhibited only a single monomer peak. In addition, dimer formation was also demonstrated in a chemical cross-linking experiment using HeLa cells transfected with 52alpha. To evaluate effects on transcription, eukaryotic expression plasmids encoding 52alpha or 52beta fused with the GAL4 DNA binding (DB) domain were co-transfected into 293 cells together with a luciferase reporter vector. A 6-fold increase in transcription activity of the reporter was detected with the GAL4-DB-52beta fusion constructs compared to GAL4-DB-52alpha or the empty vector control. We speculate that the ratio of cellular 52alpha and 52beta may play an important role in regulating gene expression as potential repressor and activator respectively.
Wang
D
D
Department of Molecular and Experimental Medicine, DNA Core Laboratory for Structural Analysis, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Buyon
J P
JP
Yang
Z
Z
Di Donato
F
F
Miranda-Carus
M E
ME
Chan
E K
EK
D001324:Autoantigens / Q000737:chemistry* / Q000235:genetics; D002850:Chromatography, Gel; D018076:DNA, Complementary / Q000737:chemistry; D019281:Dimerization; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D016350:Leucine Zippers; D008156:Luciferases / Q000235:genetics; D020733:RNA, Small Cytoplasmic; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D020798:Two-Hybrid System Techniques; D001616:beta-Galactosidase / Q000032:analysis; D000097585:SS-B Antigen; D000099207:SS-A Antigen
D001324:Autoantigens; D018076:DNA, Complementary; D008156:Luciferases; D020733:RNA, Small Cytoplasmic; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D001616:beta-Galactosidase; D000097585:SS-B Antigen; D000099207:SS-A Antigen; C116006:RO60 protein, human