Ghosh S, Jain A, Mukherjee B, Habib S, Hasnain S E
Eukaryotic Gene Expression Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
J Virol. 1998 Sep;72(9):7484-93. doi: 10.1128/JVI.72.9.7484-7493.1998.
Hypertranscription and temporal expression from the Autographa californica nuclear polyhedrosis (AcNPV) baculovirus polyhedrin promoter involves an alpha-amanitin-resistant RNA polymerase and requires a trans-acting viral factor(s). We previously reported that a 30-kDa host factor, polyhedrin promoter binding protein (PPBP), binds with unusual affinity, specificity, and stability to the transcriptionally important motif AATAAATAAGTATT within the polyhedrin (polh) initiator promoter and also displays coding strand-specific single-stranded DNA (ssDNA)-binding activity (S. Burma, B. Mukherjee, A. Jain, S. Habib, and S. E. Hasnain, J. Biol. Chem. 269:2750-2757, 1994; B. Mukherjee, S. Burma, and S. E. Hasnain, J. Biol. Chem. 270:4405-4411, 1995). We now present evidence which indicates that an additional factor(s) is involved in stabilizing PPBP-duplex promoter and PPBP-ssDNA interactions. TBP (TATA box binding protein) present in Spodoptera frugiperda (Sf9) cells is characteristically distinct from PPBP and does not interact directly with the polh promoter. Replacement of PPBP cognate sequences within the polh promoter with random nucleotides abolished PPBP binding in vitro and also failed to express the luciferase reporter gene in vivo. Phosphocellulose fractions of total nuclear extract from virus-infected cells which support in vitro transcription from the polh promoter contain PPBP activity. When PPBP was sequestered by the presence of oligonucleotides containing PPBP cognate sequence motifs, in vitro transcription of a C-free reporter cassette was affected but was restored by the exogenous addition of nuclear extract containing PPBP. When PPBP was mopped out in vivo by a plasmid carrying PPBP cognate sequence present in trans, polh promoter-driven expression of the luciferase reporter was abolished, demonstrating that binding of PPBP to the polh promoter is essential for transcription.
苜蓿银纹夜蛾核型多角体病毒（AcNPV）杆状病毒多角体蛋白启动子的超转录和时序表达涉及一种对α-鹅膏蕈碱耐药的RNA聚合酶，并且需要一种反式作用病毒因子。我们之前报道过一种30 kDa的宿主因子，多角体蛋白启动子结合蛋白（PPBP），它以不同寻常的亲和力、特异性和稳定性与多角体（polh）起始启动子内转录重要基序AATAAATAAGTATT结合，并且还表现出编码链特异性单链DNA（ssDNA）结合活性（S. 缅甸、B. 穆克吉、A. 贾因、S. 哈比卜和S. E. 哈桑奈因，《生物化学杂志》269:2750 - 2757，1994；B. 穆克吉、S. 缅甸和S. E. 哈桑奈因，《生物化学杂志》270:4405 - 4411，1995）。我们现在提供的证据表明，还有其他因子参与稳定PPBP - 双链启动子和PPBP - ssDNA相互作用。草地贪夜蛾（Sf9）细胞中存在 的TBP（TATA盒结合蛋白）与PPBP具有特征性差异，并且不直接与polh启动子相互作用。用随机核苷酸取代polh启动子内的PPBP同源序列，在体外消除了PPBP结合，并且在体内也未能表达荧光素酶报告基因。来自病毒感染细胞的总核提取物的磷酸纤维素级分支持polh启动子的体外转录，其中含有PPBP活性。当PPBP被含有PPBP同源序列基序的寡核苷酸存在所隔离时，不含C的报告盒的体外转录受到影响，但通过外源添加含有PPBP的核提取物得以恢复。当PPBP在体内被携带反式存在的PPBP同源序列的质粒清除时，荧光素酶报告基因的polh启动子驱动表达被消除，表明PPBP与polh启动子的结合对转录至关重要。
