THE ENHANCER FACTOR-R OF EPSTEIN-BARR-VIRUS (EBV) IS A SEQUENCE-SPECIFIC DNA-BINDING PROTEIN
THE ENHANCER FACTOR-R OF EPSTEIN-BARR-VIRUS (EBV) IS A SEQUENCE-SPECIFIC DNA-BINDING PROTEIN
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DOI：
10.1093/nar/18.23.6835
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发表时间：
1990-12-11
影响因子：
14.9
通讯作者：
SERGEANT, A
中科院分区：
文献类型：
--
作者：
GRUFFAT, H;MANET, E;SERGEANT, A
在潜伏感染EBV的细胞中，从潜伏感染到生产性感染的转变与两种称为EB 1（或Z）和R的EBV转录因子的表达有关。EB 1是与AP 1/ATF家族具有部分同源性的上游元件因子，而R是增强子因子。 在仅在EBV裂解周期期间有活性的复制起点的R-应答增强子（ORllyt）中，R-应答元件位于约70 bp的区域（RRE-DR）。在这里，我们表明，R，无论是在体外翻译，或存在于HeLa细胞组成性生产R的核提取物，直接结合和保护对DNAase II消化，两个区域在RRE-DR。使用迁移率变动分析和DMS干扰，我们的特点是R和DNA之间的接触点。 RRE-DR中有两个相邻的结合位点RRE-DR 1和RRE-DR 2，RRE-DR 1和RRE-DR 2中分别相隔7 bp和6 bp的两个序列cctGTGCCttgtcccGTGGACaatgtccc和caatGTCCCtccagcGTGGTGgctg。 R与其同源序列的直接相互作用由其N-末端355个氨基酸赋予。 在RRE-DR中的定向诱变，无论是R-结合位点，R在体外的结合受损，并通过在HeLa细胞中的瞬时表达测定，受损的R-激活的两个因素。 这表明RRE-DR 1和RRE-DR 2对R的反应不协同。
In cells latently infected with EBV, the switch from latency to productive infection is linked to the expression of two EBV transcription factors called EB1 (or Z) and R. EB1 is an upstream element factor which has partial homology to the AP1/ATF family, whereas R is an enhancer factor. In the R-responsive enhancer of the replication origin only active during the EBV lytic cycle (ORllyt), R-responsive elements are located in a region of about 70 bp (RRE-DR). Here we show that R, produced either by in vitro translation, or present in nuclear extracts from HeLa cells constitutively producing R, binds directly to and protects against DNAase Il digestion, two regions in RRE-DR. Using mobility shift assay and DMS interference, we have characterized the contact-points between R and the DNA. Two binding sites, RRE-DR1 and RRE-DR2, were characterized and are contiguous in RRE-DR. R binds to theses two sites probably by simultaneously contacting two sequences within the sites, which are separated by 7 bp in RRE-DR1, cctGTGCCttgtcccGTGGACaatgtccc, and by 6bp in RRE-DR2, caatGTCCCtccagcGTGGTGgctg. Direct interaction of R with its cognate sequences is conferred by its N-terminal 355 amino-acids. Directed mutagenesis in RRE-DR, of either R-binding site, impaired binding of R in vitro and, as assayed by transient expression in HeLa cells, impaired R-activation by a factor of two. This suggests that RRE-DR1 and RRE-DR2 do not respond cooperatively to R.