CHARACTERIZATION OF THE DNA-BINDING SITE REPERTOIRE FOR THE EPSTEIN-BARR-VIRUS TRANSCRIPTION FACTOR-R

The Epstein-Barr virus gene BRLF1 encodes the transcription factor R, which is a sequence-specific DNA-binding protein important for the swi tch from latency to a productive cycle. We have defined a repertoire o f specific R-binding sites using a GST-R fusion protein and a pool of 23 bp random DNA sequences. The R-bound sequences were selected by sev eral rounds of Electrophoretic Mobility Shift Assay (EMSA) and amplifi cation by PCR. Among the 45 sites selected, some positions in the sequ ences were highly conserved, i.e., 5'-G(T)GCC N(7)GTGGTG-3'. The guani ne methylation assay revealed that R simultaneously contacts guanines in the two conserved cores, defining the consensus binding site 5'-GNC C N-9 GGNG-3', and 30 sites among the 45 selected have this sequence. This last result also suggests that R binds two adjacent major grooves of the DNA. As shown by EMSA assay, R binds to all the sites tested w ith a comparable affinity, and they all mediate R-induced transcriptio nal activation in a transient expression assay.