Binding of the pseudorabies virus immediate-early protein to single-stranded DNA.
Binding of the pseudorabies virus immediate-early protein to single-stranded DNA.
复制标题
伪狂犬病病毒立即早期蛋白与单链 DNA 的结合。
DOI：
10.1128/jvi.61.6.1855-1860.1987
复制
发表时间：
1987
影响因子：
5.4
通讯作者：
Feldman,LT
中科院分区：
文献类型：
--
作者：
Chlan,CA;Coulter,C;Feldman,LT
为了将激活转录的能力与对单链DNA的亲和力相关联，测试了野生型和温度敏感性伪狂犬病病毒立即早期蛋白与单链DNA柱结合的能力。野生型和温度敏感的立即早期蛋白质在0 ℃和40 ℃下以相似的亲和力与非特异性单链DNA柱结合。在激活转录的能力和结合单链DNA的能力之间似乎没有直接的相关性。为了进一步研究与单链DNA结合的相互作用，我们在大肠杆菌表达载体中表达了立即早期蛋白。在该系统中，表达的立即早期蛋白质不被磷酸化，也不能与哺乳动物细胞因子复合。第一个trp构建体不表达可溶形式的立即早期蛋白，推测是由于trp前导序列的不溶性。我们删除了一大段trpE基因，发现立即早期融合蛋白是可溶性的。我们通过单链DNA纤维素柱测试了这种蛋白质对单链DNA的亲和力。细菌表达的立即早期蛋白结合单链DNA至少和野生型蛋白一样好。单链DNA的亲和力似乎不依赖于磷酸化状态，也不依赖于哺乳动物细胞因子的存在。
In an attempt to correlate the ability to activate transcription with affinity for single-stranded DNA, both wild-type and temperature-sensitive pseudorabies virus immediate-early proteins were tested for the ability to bind to single-stranded DNA columns. Wild-type and temperature-sensitive immediate-early proteins bound to nonspecific single-stranded DNA columns with similar affinities at both 0 and 40 degrees C. There did not seem to be a direct correlation between the ability to activate transcription and the ability to bind to single-stranded DNA. To study further the interactions that are involved in binding to single-stranded DNA, we expressed the immediate-early protein in an Escherichia coli expression vector. In this system the expressed immediate-early protein was not phosphorylated, nor could it be complexed with mammalian cell factors. The first trp construct did not express a soluble form of the immediate-early protein, presumably due to the insoluble nature of the trp leader. We deleted a large segment of the trpE gene and found that the immediate-early fusion protein was soluble. We tested this protein for its affinity for single-stranded DNA by passage over single-stranded DNA cellulose columns. The bacterially expressed immediate-early protein bound single-stranded DNA at least as well as did the wild-type protein. Affinity for single-stranded DNA did not appear to be dependent on the phosphorylation state nor on the presence of mammalian cell factors.