Figure 5

EBNA1 bound to FR impedes transcription fork progression. (A) Representation of transcription reporter plasmids used here. In pRSVL, the RSV LTR drives transcription of the luciferase gene, and the SV40 late polyadenylation signal is used for polyadenylation. Derivatives of pRSVL with ten, 20 or 40 EBNA1-binding sites (filled black circles) between the luciferase gene and the polyadenylation signal were constructed. Primary transcripts, and prematurely terminated transcripts present in total RNA preparations are indicated, as are mature luciferase mRNAs present in total and polyA+ RNA preparations. Primers used for RT-PCR are indicated as arrows. (B) Luciferase expression from the reporter plasmids described above. Plasmids were co-transfected with either pcDNA3 (stippled bars), or a EBNA1 DNA binding domain expression plasmid (black bars) into 293 cells. The number of EBNA1 binding sites in the reporter plasmid is indicated below each pair of bars. Luciferase activity is reported relative to the activity observed when pRSVL was co-transfected with pcDNA3. (C) RT-PCRs to detect luciferase and GAPDH transcripts in total or polyadenylated RNAs recovered from the transfected cells described in B. PCR products were visualized with ethidium bromide and the identity of the transfected plasmid is indicated above each lane.