AcMNPV late expression factor-5 interacts with itself and contains a zinc ribbon domain that is required for maximal late transcription activity and is homologous to elongation factor TFIIS.
AcMNPV late expression factor-5 interacts with itself and contains a zinc ribbon domain that is required for maximal late transcription activity and is homologous to elongation factor TFIIS.
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AcMNPV 晚期表达因子 5 与其自身相互作用，并包含最大晚期转录活性所需的锌带结构域，并且与延伸因子 TFIIS 同源。
DOI：
10.1006/viro.1998.9334
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Rohrmann,GF
中科院分区：
文献类型：
--
作者：
Harwood,SH;Li,L;Ho,PS;Preston,AK;Rohrmann,GF
苜蓿银纹夜蛾核型多角体病毒（Autographa californicamultinucleocapsidpolyhedrovirus，AcMNPV）晚期表达因子5（lef-5）基因是晚期基因表达所必需的。在本文中，我们证明了LEF-5在酵母双杂交系统和谷胱甘肽-S-转移酶亲和力测定中与自身相互作用。缺失分析表明，C-末端71个氨基酸（aa）的相互作用是不需要的。然而，所有涉及N-末端194 aa的缺失均显著降低了LEF-5：LEF-5相互作用。将LEF-5或LEF-5缺失突变体与杆状病毒晚期转录所需的基因的完整互补物一起转染到Sf-9细胞中。所有缺失克隆都检测到在latevp 39衣壳启动子控制下的β-葡萄糖醛酸酶（GUS）报告基因的表达降低。LEF-5的氨基酸序列分析确定了一个以前未报道的结构域内的C-末端32个氨基酸是同源的RNA聚合酶II延伸因子IIS（TFIIS）的锌带结构域从各种类群。使用TFIIS的Zn带的NMR数据对推定的LEF-5 Zn带进行分子建模，表明该域可以折叠成类似于TFIIS的Zn带结构。在瞬时表达测定中，预测对LEF-5带状结构的功能重要的氨基酸的丙氨酸扫描诱变显著降低了LEF-5活性。突变改变的氨基酸预测协调锌+引起的活性降低类似的域被完全消除时。
The late expression factor-5 gene (lef-5) ofAutographa californicamultinucleocapsid polyhedrovirus (AcMNPV) is required for late gene expression. In this paper, we demonstrate that LEF-5 interacts with itself in the yeast two-hybrid system and in glutathione–S-transferase affinity assays. Deletion analysis suggested that the C-terminal 71 amino acids (aa) were not required for interaction. However, all deletions tested involving the N-terminal 194 aa significantly reduced LEF-5:LEF-5 interaction. LEF-5 or LEF-5 deletion mutants were transfected into Sf-9 cells with the full complement of genes required for baculovirus late transcription. All deletion clones tested reduced expression of a β-glucuronidase (GUS) reporter gene under control of the latevp39capsid promoter. Amino-acid sequence analysis of LEF-5 identified a previously unreported domain within the C-terminal 32 aa that is homologous to the zinc ribbon domain of RNA polymerase II elongation factor IIS (TFIIS) from a variety of taxa. Molecular modeling of the putative LEF-5 Zn ribbon using the NMR data available for the Zn ribbon of TFIIS suggested that this domain could fold into a Zn ribbon structure similar to TFIIS. Alanine scanning mutagenesis of amino acids predicted to be important for functioning of the LEF-5 ribbon structure significantly reduced LEF-5 activity in transient expression assays. Mutations changing the amino acids predicted to coordinate Zn2+caused a reduction in activity similar to that when the domain was eliminated completely.