Nucleic Acids Research, 1994, Vol. 22, No. 7 1172-1178
© 1994
MOLECULAR BIOLOGY |
Characterization of the DNA-binding site repertoire for the Epstein - Barr virus transcription factor R
U 412 INSERM, Unité de Virologie Humaine de L'Ecole Normale Supérieure de Lyon 46 Allée d'ltalie, 69364 Lyon Cedex 07, France
*To whom correspondence should be addressed
Received January 4, 1994. Revised February 23, 1994. Accepted February 23, 1994.
The Epstein-Barr virus gene BRLF1 encodes the transcription factor R, which is a sequence-specific DNA-binding protein important for the switch from latency to a productive cycle. We have defined a repertoire of specific R-bindlng sites using a GST-R fusion protein and a pool of 23 bp random DNA sequences. The R-bound sequences were selected by several rounds of Electrophoretic Mobility Shift Assay (EMSA) and amplification by PCR. Among the 45 sites selected, some positions in the sequences were highly conserved, i.e., 5'-GTGCC N9GTGGTG-3'. The guanine methylation assay revealed that R simultaneously contacts guanines in the two conserved cores, defining the consensus binding site 5'-GNCC N9GGNG-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 with a comparable affinity, and they all mediate R-induced transcriptional activation in a transient expression assay.
+Present address: Instirut für Klinische Molekularbiologie und Tumorgenetik. GSF-Forschungszentrum für Umwell und Gesundheit. GmbH Marchioninistrasse 25. D-81377 Munich, Germany
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