DNA-binding site of major regulatory protein alpha 4 specifically associated with promoter-regulatory domains of alpha genes of herpes simplex virus type 1.
DNA-binding site of major regulatory protein alpha 4 specifically associated with promoter-regulatory domains of alpha genes of herpes simplex virus type 1.
复制标题
DOI：
10.1073/pnas.83.13.4700
复制
发表时间：
1986-07
影响因子：
11.1
通讯作者：
T. Kristie;B. Roizman
中科院分区：
文献类型：
--
作者：
T. Kristie;B. Roizman
单纯疱疹病毒1型基因形成至少五组（α、β 1、β 2、γ 1和γ 2），其表达以级联方式协调调节并顺序排列。以前的研究表明，功能性α 4基因产物是从α蛋白合成到β蛋白合成的过渡所必需的，并表明α 4基因表达是自动调节的。我们以前曾报道，标记的DNA片段含有启动子调控结构域的三个α（α 0，α 4，和α 27）和γ 2基因形成稳定的复合物与蛋白质从裂解物的感染细胞检测到的凝胶电泳结合试验和单克隆抗体α 4蛋白降低电泳迁移率的标记的DNA和蛋白质从感染细胞。在这项研究中，我们确定了一个单克隆抗体，H950，从一组单克隆抗体的α 4蛋白，阻止特定的感染细胞复合物的形成与标记的DNA片段含有启动子和调节结构域的α基因。我们还报告了在不存在H950单克隆抗体的情况下，但在其存在下，α 4蛋白质保护α 0启动子结构域免受核酸外切酶III消化的核苷酸序列。此外，我们还从α 4基因的调节结构域中鉴定了一个59个碱基对的序列，该序列与α 4蛋白结合。该片段的缺失克隆定位形成α 4蛋白-DNA复合物所需的序列元件。此外，从59个碱基对片段中删除SP1转录因子的体外结合位点并不影响α 4蛋白-DNA复合物的形成。
Herpes simplex virus type 1 genes form at least five groups (alpha, beta 1, beta 2, gamma 1, and gamma 2) whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Previous studies have shown that functional alpha 4 gene product is essential for the transition from alpha to beta protein synthesis and have suggested that alpha 4 gene expression is autoregulatory. We have previously reported that labeled DNA fragments containing promoter-regulatory domains of three alpha (alpha 0, alpha 4, and alpha 27) and a gamma 2 gene form stable complexes with proteins from lysates of infected cells as detected by a gel electrophoresis binding assay and that monoclonal antibody to alpha 4 protein reduced the electrophoretic mobility of the complex of labeled DNA and protein from infected cells. In this study we identified one monoclonal antibody, H950, from a panel of monoclonal antibodies to the alpha 4 protein that blocks the formation of specific infected cell complexes with labeled DNA fragments containing promoter and regulatory domains of alpha genes. We also report the nucleotide sequence of the alpha 0 promoter domain protected from exonuclease III digestion by alpha 4 protein in the absence of H950 monoclonal antibody but not in its presence. In addition, we identified a 59-base-pair sequence from the regulatory domain of the alpha 4 gene that binds alpha 4 protein. Deletion clones of this fragment localize sequence elements required for formation of the alpha 4 protein-DNA complex. Furthermore, deletion of the in vitro binding site of the SP1 transcription factor from the 59-base-pair fragment did not affect the formation of the alpha 4 protein-DNA complex.