CHARACTERIZATION OF THE DNA-BINDING SITE REPERTOIRE FOR THE EPSTEIN-BARR-VIRUS TRANSCRIPTION FACTOR-R
CHARACTERIZATION OF THE DNA-BINDING SITE REPERTOIRE FOR THE EPSTEIN-BARR-VIRUS TRANSCRIPTION FACTOR-R
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DOI：
10.1093/nar/22.7.1172
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发表时间：
1994-04-11
影响因子：
14.9
通讯作者：
SERGEANT, A
中科院分区：
文献类型：
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作者：
GRUFFAT, H;SERGEANT, A
Epstein-Barr病毒基因BRLF1编码转录因子R，这是一种序列特异的DNA结合蛋白，对于从潜伏到生产周期的转换非常重要。我们使用GST-R融合蛋白和23bp的随机DNA序列定义了一系列特定的R结合位点。经多轮凝胶迁移率漂移分析(EMSA)和聚合酶链式反应(PCR)扩增，筛选出R结合序列。在所选择的45个位点中，序列中的一些位置高度保守，即5‘-G(T)GCC N(7)GTGGTG-3’。鸟嘌呤甲基化分析表明，R同时与两个保守核心中的鸟嘌呤接触，定义了共同的结合位点5‘-GNCC N-9 GGNG-3’，在所选的45个位点中有30个具有该序列。这最后的结果也表明R结合了DNA的两个相邻的主槽。EMSA分析表明，R以相似的亲和力结合到所有被测试的位点，并且它们都在瞬时表达实验中介导了R诱导的转录激活。
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-binding 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'-G(T)GCC N(7)GTGGTG-3'. The guanine methylation assay revealed that R simultaneously contacts guanines in the two conserved cores, defining the consensus binding site 5'-GNCC 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 with a comparable affinity, and they all mediate R-induced transcriptional activation in a transient expression assay.