Figure 5. UR1-mediated self-interaction is required for EBNA1 to transactivate cooperatively.

(A) Size-exclusion chromatography of the wild-type UR1 peptide indicates zinc-dependent dimerization. WT peptide was subjected to HPLC size-exclusion chromatography, as described in the Methods section, in the absence of zinc (red), or in the presence of 1 mM zinc sulfate in the buffer (green). The retention time is indicated above each peak, and the apparent molecular weight calculated from the retention time is shown in parentheses. The elution profile of molecular weight markers is shown in gray. The known molecular weight of each marker is indicated above the peak, with the observed retention time shown in parentheses. (B) Cooperative transactivation by EBNA1 is UR1-dependent. C33a cells were co-transfected individually with TKp-luciferase reporter plasmids containing one, three, five, seven, ten, or 20 EBNA1 binding sites in FR along with an expression plasmid for EBNA1 or EBNA1(CC→SS). Cells were harvested at 48 hours post-transfection, normalized by flow cytometry for the number of live-transfected cells, and analyzed for luciferase activity, which is expressed as luciferase activity fold-over the activity obtained by co-transfection with vector control. The white bars indicate the predicted levels of transactivation for EBNA1 if it increased additively with increasing numbers of binding sites. The transactivation observed with EBNA1 is indicated by the black bars, and transactivation observed with EBNA1(CC→SS) is indicated by the gray bars. For reporter plasmids containing up to three binding sites, EBNA1 and EBNA1(CC→SS) transactivated equivalently. A small difference was observed for a reporter plasmid with five binding sites, and this difference was greatly accentuated for reporter plasmids containing seven, ten, and 20 binding sites. (C) Cooperative transactivation by EBNA1 fits a sigmoidal dose-response model by non-linear regression analysis. The fold transactivation by EBNA1 observed in (5B) was analyzed for cooperativity as described in the Materials & Methods, and found to fit a sigmoidal dose-response model well, as indicated by the goodness of fit (R²). Positive-cooperativity is inferred from the positive Hill coefficient (HC). (D) EBNA1(CC→SS) does not activate transcription cooperatively. The fold transactivation observed for EBNA1(CC→SS) was fit to the same sigmoidal dose-response model used in 5C. The low R² indicates that transactivation by EBNA1(CC→SS) poorly fits this model. In addition, the large standard deviation observed for the HC also indicates a lack of cooperative transactivation.