Kagaya Y, Ohmiya K, Hattori T
Center for Molecular Biology and Genetics and Department of Bioresources, School of Bioresources, Mie University, 1515 Kamihama-cho, Tsu 514-8507, Japan.
Nucleic Acids Res. 1999 Jan 15;27(2):470-8. doi: 10.1093/nar/27.2.470.
We have cloned and characterized two novel DNA binding proteins designated RAV1 and RAV2 from Arabidopsis thaliana. RAV1 and RAV2 contain two distinct amino acid sequence domains found only in higher plant species. The N-terminal regions of RAV1 and RAV2 are homologous to the AP2 DNA-binding domain present in a family of transcription factors represented by the Arabidopsis APETALA2 and tobacco EREBP proteins, while the C-terminal region exhibits homology to the highly conserved C-terminal domain, designated B3, of VP1/ABI3 transcription factors. Binding site selection assays using a recombinant glutathione S-transferase fusion protein have revealed that RAV1 binds specifically to bipartite recognition sequences composed of two unrelated motifs, 5'-CAACA-3' and 5'-CACCTG-3', separated by various spacings in two different relative orientations. Analyses using various deletion derivatives of the RAV1 fusion protein show that the AP2 and B3-like domains of RAV1 bind autonomously to the CAACA and CACCTG motifs, respectively, and together achieve a high affinity and specificity of binding. From these results, we suggest that the AP2 and B3-like domains of RAV1 are connected by a highly flexible structure enabling the two domains to bind to the CAACA and CACCTG motifs in various spacings and orientations.
我们从拟南芥中克隆并鉴定了两种新型DNA结合蛋白，分别命名为RAV1和RAV2。RAV1和RAV2含有仅在高等植物物种中发现的两个不同的氨基酸序列结构域。RAV1和RAV2的N端区域与以拟南芥APETALA2和烟草EREBP蛋白为代表的转录因子家族中存在的AP2 DNA结合结构域同源，而C端区域与VP1/ABI3转录因子高度保守的C端结构域（称为B3）具有同源性。使用重组谷胱甘肽S-转移酶融合蛋白进行的结合位点选择试验表明，RAV1特异性结合由两个不相关基序5'-CAACA-3'和5'-CACCTG-3'组成的双部分识别序列，这两个基序在两种不同的相对方向上被不同间距隔开。使用RAV1融合蛋白的各种缺失衍生物进行的分析表明，RAV1的AP2和B3样结构域分别自主结合CAACA和CACCTG基序，并共同实现高亲和力和特异性结合。从这些结果来看，我们认为RAV1的AP2和B3样结构域通过高度灵活的结构相连，使这两个结构域能够以各种间距和方向结合CAACA和CACCTG基序。
