McBroom L D, Flock G, Giguère V
Department of Biochemistry, McGill University, Royal Victoria Hospital, Montréal, Québec, Canada.
Mol Cell Biol. 1995 Feb;15(2):796-808. doi: 10.1128/MCB.15.2.796.
ROR alpha 1 and ROR alpha 2 are two isoforms of a novel member of the steroid-thyroid-retinoid receptor superfamily and are considered orphan receptors since their cognate ligand has yet to be identified. These putative receptors have previously been shown to bind as monomers to a DNA recognition sequence composed of two distinct moieties, a 3' nuclear receptor core half-site AGGTCA preceded by a 5' AT-rich sequence. Recognition of this bipartite hormone response element (RORE) requires both the zinc-binding motifs and a group of amino acid residues located at the carboxy-terminal end of the DNA-binding domain (DBD) which is referred to here as the carboxy-terminal extension. In this report, we show that binding of ROR alpha 1 and ROR alpha 2 to the RORE induces a large DNA bend of approximately 130 degrees which may be important for receptor function. The overall direction of the DNA bend is towards the major groove at the center of the 3' AGGTCA half-site. The presence of the nonconserved hinge region which is located between the DBD and the putative ligand-binding domain (LBD) or ROR alpha is required for maximal DNA bending. Deletion of a large portion of the amino-terminal domain (NTD) of the ROR alpha protein does not alter the DNA bend angle but shifts the DNA bend center 5' relative to the bend induced by intact ROR alpha. Methylation interference studies using the NTD-deleted ROR alpha 1 mutant indicate that some DNA contacts in the 5' AT-rich half of the RORE are also shifted 5', while those in the 3' AGGTCA half-site are unaffected. These results are consistent with a model in which the ROR alpha NTD and the nonconserved hinge region orient the zinc-binding motifs and the carboxy-terminal extension of the ROR alpha DBD relative to each other to achieve proper interactions with the two halves of its recognition site. Transactivation studies suggest that both protein-induced DNA bending and protein-protein interactions are important for receptor function.
RORα1和RORα2是类固醇-甲状腺-视黄酸受体超家族一个新成员的两种亚型，由于其同源配体尚未确定，它们被视为孤儿受体。此前已表明，这些假定的受体以单体形式结合到一个由两个不同部分组成的DNA识别序列上，一个是3'核受体核心半位点AGGTCA，前面是一个5'富含AT的序列。对这种二分体激素反应元件（RORE）的识别既需要锌结合基序，也需要位于DNA结合结构域（DBD）羧基末端的一组氨基酸残基，此处将其称为羧基末端延伸。在本报告中，我们表明RORα1和RORα2与RORE的结合会诱导大约130度的大DNA弯曲，这可能对受体功能很重要。DNA弯曲的总体方向是朝向3' AGGTCA半位点中心的大沟。位于DBD和假定的配体结合结构域（LBD）之间的非保守铰链区或RORα的存在是最大程度DNA弯曲所必需的。RORα蛋白氨基末端结构域（NTD）的大部分缺失不会改变DNA弯曲角度，但会使DNA弯曲中心相对于完整RORα诱导的弯曲向5'端移动。使用NTD缺失的RORα1突变体进行的甲基化干扰研究表明，RORE的5'富含AT的一半中的一些DNA接触点也向5'端移动，而3' AGGTCA半位点中的接触点则不受影响。这些结果与一个模型一致，在该模型中，RORα NTD和非保守铰链区使RORα DBD的锌结合基序和羧基末端延伸相互定向，以实现与其识别位点的两半进行适当的相互作用。反式激活研究表明，蛋白质诱导的DNA弯曲和蛋白质-蛋白质相互作用对受体功能都很重要。
