Mapping the RNA-binding domain on the Apple chlorotic leaf spot virus movement protein
Mapping the RNA-binding domain on the Apple chlorotic leaf spot virus movement protein
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DOI：
10.1099/vir.0.80493-0
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发表时间：
2005-01-01
影响因子：
3.8
通讯作者：
Yoshikawa, N
中科院分区：
文献类型：
--
作者：
Isogai, M;Yoshikawa, N
分析了苹果褪绿叶斑病毒细胞间运动蛋白（MP）的RNA结合特性。IMP在大肠杆菌中表达，并用于UV交联分析，使用地高辛-UTP标记的RNA探针和凝胶阻滞分析。分析表明MP与单链RNA（ssRNA）协同结合。当分析RNA结合活性的NaCl依赖性时，即使在含有1 M NaCl的结合缓冲液中，大多数IMP也可以结合ssRNA。此外，竞争结合实验表明，MP优先绑定到ssRNA和单链DNA没有序列特异性。MP缺失突变体用于通过UV交联分析鉴定RNA结合结构域。氨基酸残基82-126和127-287可能含有两个独立活性的单链核酸结合结构域。
The RNA-binding properties of the cell-to-cell movement protein (MP) of Apple chlorotic leaf spot virus were analysed. IMP was expressed in Escherichia coli and was used in UV-crosslinking analysis, using a digoxigenin-UTP-labelled RNA probe and gel-retardation analysis. The analyses demonstrated that MP bound cooperatively to single-stranded RNA (ssRNA). When analysed for NaCl dependence of the RNA-binding activity, the majority of the IMP could bind ssRNA even in binding buffer with 1 M NaCl. Furthermore, competition binding experiments showed that the MP bound preferentially to ssRNA and single-stranded DNA without sequence specificity. MP deletion mutants were used to identify the RNA-binding domain by UV-crosslinking analysis. Amino acid residues 82-126 and 127-287 potentially contain two independently active, single-stranded nucleic acid-binding domains.