Ishiguro S, Tanaka M, Kojimoto A, Kato M, Iwabuchi M, Nakamura K
Laboratory of Biochemistry, School of Agriculture, Nagoya University, Japan.
Plant Cell Physiol. 1993 Jun;34(4):567-76.
A nuclear extract from petioles of sweet potato protected several sites in the 5'-upstream region of a gene for beta-amylase from DNase I digestion. One of these sites, located at a region around 800-base pairs upstream from the transcription start site, having an imperfect palindromic sequence of CGTCACGTCACG, was designated the R-box. The site contained tandemly duplicated CGTCA sequences, referred to below as 5'- and 3'-CGTCA. Competition experiments in gel mobility shift assays with mutant R-box oligonucleotides indicated that mutations in bases outside the 3'-CGTCA of the R-box do not severely affect the binding. By contrast, single-base substitutions in any one base of the 3'-CGTCA greatly abolished the binding even when the mutated R-boxes contained intact 5'-CGTCA. However, oligonucleotides with mutations in the 3'-CGTCA had the ability to bind the nuclear factor when additional mutations were introduced to create a partially palindromic sequence containing the CGTCA sequence in its 3'-half on the opposite strand. These results indicate that the CGTCA sequence alone is not sufficient for the binding of the R-box binding factor (RBF) and that the RBF binds to the sequence with partial dyad symmetry that contains the CGTCA motif in its 3'-half. The optimum sequence for the binding of the RBF is suggested to be a palindromic octameric sequence TGACGTCA, which is identical to the consensus sequence of the cAMP-responsive element (CRE) of animal genes. Bacterially produced HBP-1b of wheat bound to the R-box, and its binding to mutated R-boxes was similar to that of RBF, suggesting that the RBF belongs to a family of bZIP-type plant nuclear factors that bind to CGTCA-related sequences. However, several differences between the RBF and HBP-1b were also noted.
甘薯叶柄的核提取物可保护β-淀粉酶基因5'-上游区域的几个位点不被DNase I消化。其中一个位点位于转录起始位点上游约800个碱基对处，具有CGTCACGTCACG的不完全回文序列，被命名为R-box。该位点包含串联重复的CGTCA序列，以下称为5'-和3'-CGTCA。用突变的R-box寡核苷酸进行凝胶迁移率变动分析的竞争实验表明，R-box的3'-CGTCA以外的碱基突变不会严重影响结合。相比之下，3'-CGTCA中任何一个碱基的单碱基替换即使在突变的R-box包含完整的5'-CGTCA时也会大大消除结合。然而，如果在3'-CGTCA中引入额外突变以在其互补链的3'-半部分创建包含CGTCA序列的部分回文序列，则具有3'-CGTCA突变的寡核苷酸仍具有结合核因子的能力。这些结果表明，单独CGTCA序列不足以与R-box结合因子（RBF）结合，并且RBF与具有部分二重对称的序列结合，该序列在其3'-半部分包含CGTCA基序。RBF结合的最佳序列被认为是回文八聚体序列TGACGTCA，它与动物基因的cAMP反应元件（CRE）的共有序列相同。小麦细菌产生的HBP-1b与R-box结合，其与突变R-box的结合与RBF相似，表明RBF属于与CGTCA相关序列结合的bZIP型植物核因子家族。然而，也注意到RBF和HBP-1b之间的一些差异。
