Schindler U, Terzaghi W, Beckmann H, Kadesch T, Cashmore A R
Plant Science Institute, University of Pennsylvania, Philadelphia 19104.
EMBO J. 1992 Apr;11(4):1275-89. doi: 10.1002/j.1460-2075.1992.tb05171.x.
The G-box is a cis-acting element found within the promoters of many plant genes where it mediates expression in response to a variety of different stimuli. This palindromic DNA motif (CCACGTGG) is composed of two identical half sites, the base pairs of which we have numbered -4 to +4 (numbering from 5' to 3'). Both half sites are involved in the binding of the bZIP protein GBF1, a member of the GBF family of Arabidopsis thaliana. Here we demonstrate using the random binding site selection method that GBF1 interacts with, in addition to the palindromic G-box, other DNA motifs that fall into seven distinct groups. All groups share the ACGT core sequence, common to most DNA motifs bound by plant bZIP proteins so far characterized. Our studies demonstrate that a high affinity GBF1 binding site is further defined by the following two parameters: first, all sites contain a G residue at position +3 (as in ACGTG) and secondly, only certain base pair combinations are allowed at positions -4, -3 and +4. Two of the identified groups (TGACGTGG and TGACGTGT) contain the base pairs TG at positions -4 and -3 and hence resemble the binding sites of another class of plant bZIP proteins (TGACGT/C binding proteins). However, GBF1 only interacts with the TGACGT sequence if the two 3' distal nucleotides (positions +3 and +4) are occupied by GG or GT. These data define the differences between a G-box binding protein and TGACGT/C binding proteins. The N-terminal domain of GBF1 is defined by a high proline content. Such regions were also identified in proteins related to GBF1. We demonstrate that this N-terminal proline-rich domain of GBF1, when fused to a heterologous DNA binding domain, stimulates transcription in both plant protoplasts and mammalian cells. These extensive DNA binding studies and the characterization of the GBF1 activation domain will facilitate both the identification of regulatory elements and the in vivo function of GBF1.
G盒是一种顺式作用元件，存在于许多植物基因的启动子中，介导对多种不同刺激的响应表达。这种回文DNA基序（CCACGTGG）由两个相同的半位点组成，我们将其碱基对编号为-4至+4（从5'到3'编号）。两个半位点都参与bZIP蛋白GBF1的结合，GBF1是拟南芥GBF家族的成员。在这里，我们使用随机结合位点选择方法证明，GBF1除了与回文G盒相互作用外，还与其他分为七个不同组的DNA基序相互作用。所有组都共享ACGT核心序列，这是迄今为止已鉴定的大多数与植物bZIP蛋白结合的DNA基序所共有的。我们的研究表明，高亲和力GBF1结合位点由以下两个参数进一步定义：第一，所有位点在+3位置都含有一个G残基（如ACGTG）；第二，在-4、-3和+4位置只允许某些碱基对组合。两个已鉴定的组（TGACGTGG和TGACGTGT）在-4和-3位置含有碱基对TG，因此类似于另一类植物bZIP蛋白（TGACGT/C结合蛋白）的结合位点。然而，只有当两个3'末端核苷酸（+3和+4位置）被GG或GT占据时，GBF1才与TGACGT序列相互作用。这些数据定义了G盒结合蛋白与TGACGT/C结合蛋白之间的差异。GBF1的N末端结构域以高脯氨酸含量为特征。在与GBF1相关的蛋白质中也鉴定出了这样的区域。我们证明，GBF1的这个富含脯氨酸的N末端结构域与异源DNA结合结构域融合时，能在植物原生质体和哺乳动物细胞中刺激转录。这些广泛的DNA结合研究以及GBF1激活结构域的表征将有助于鉴定调控元件以及GBF1在体内的功能。
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