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首页> 外文期刊>Molecular and Cellular Biology >Functional Analysis of the Mad1-mSin3A Repressor-Corepressor Interaction Reveals Determinants of Specificity, Affinity, and Transcriptional Response
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Functional Analysis of the Mad1-mSin3A Repressor-Corepressor Interaction Reveals Determinants of Specificity, Affinity, and Transcriptional Response

机译：Mad1-mSin3A阻遏物-Corepressor相互作用的功能分析揭示了特异性，亲和力和转录反应的决定因素。

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The recruitment of corepressors by DNA-bound repressors is likely to be a critical rate-limiting step in the transcriptional regulation of many genes. An excellent paradigm for such an interaction is the association of the basic helix-loop-helix zipper protein Mad1 with the corepressor mSin3A. When bound together, the Sin3 interaction domain (SID) of Mad1 forms extensive hydrophobic contacts with the four-helix bundle formed by the paired amphipathic helix 2 (PAH2) domain of mSin3A. Using the costructure to predict the principle residues required for binding, we have carried out an extensive mutational analysis to examine the Mad1 SID-mSin3A PAH2 interaction in vitro and in vivo. Bulky hydrophobic residues in the α1 (I308 and V311) and α2 (L329 and L332) helices of the PAH2 domain are necessary to accommodate the precise arrangement of bulky (L12) and short (A15 and A16) hydrophobic residues in the amphipathic Mad1 SID. We have also used phage display to derive an optimal SID, which shows an essentially identical arrangement of key residues. By manipulating these key residues, we have generated altered-specificity Mad1 SID mutants that bind only to a PAH2 domain with a reciprocal mutation, permitting us to demonstrate for the first time that these domains interact directly in vivo. We have also found that the integrity of the PAH1 domain affects the Mad1 SID-PAH2 interaction. It is conceivable that cross talk between different PAH domains and their binding partners helps to determine the subunit composition and order of assembly of mSin3A complexes.
机译：DNA结合阻遏物募集corepressor可能是许多基因转录调控中关键的限速步骤。这种相互作用的一个很好的范例是基本的螺旋-环-螺旋拉链蛋白Mad1与mRNAsin3A的关联。当绑定在一起时，Mad1的Sin3相互作用域（SID）与由mSin3A的成对的两亲性螺旋2（PAH2）域形成的四螺旋束形成广泛的疏水接触。使用该结构预测结合所需的主要残基，我们进行了广泛的突变分析，以研究Mad1 SID-mSin3A PAH2在体外和体内的相互作用。 PAH2域的α1（I308和V311）和α2（L329和L332）螺旋中的大块疏水残基对于容纳两亲Mad1 SID中大块（L12）和短（A15和A16）疏水残基的精确排列是必需的。我们还使用噬菌体展示来推导最佳SID，该SID显示了关键残基的基本相同排列。通过操纵这些关键残基，我们产生了改变特异性的Mad1 SID突变体，该突变体仅与具有相互突变的PAH2域结合，这使我们首次证明了这些域在体内直接相互作用。我们还发现，PAH1域的完整性会影响Mad1 SID-PAH2相互作用。可以想象，不同的PAH域及其结合伴侣之间的相互干扰有助于确定mSin3A复合物的亚基组成和组装顺序。

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