Nurrish S J, Treisman R
Transcription Laboratory, Imperial Cancer Research Fund Laboratories, London, United Kingdom.
Mol Cell Biol. 1995 Aug;15(8):4076-85. doi: 10.1128/MCB.15.8.4076.
The MADS box is a conserved sequence motif found in the DNA binding domain of a family of transcription factors which possess related but distinct DNA binding specificities. We investigated the basis of differential sequence recognition by the MADS-box proteins serum response factor (SRF), MCM1, and MEF2A, using chimeric proteins and site-directed mutants in conjunction with gel mobility shift and binding site selection assays. Deletion of sequences immediately N terminal to the SRF MADS box alters its preferred binding site to that of MEF2A, although the resulting protein still weakly binds SRF-specific sites: exclusive binding to MEF2 sites requires further mutations, at MADS-box residues 11 to 15. In contrast to SRF, the sequence specificity of MCM1 (and of MEF2A) is determined entirely by sequences within its MADS box, and mutation of only SRF MADS-box residue 1 is sufficient to alter its binding specificity to that of MCM1. However, changes at both MADS-box positions 1 and 11 to 15 are necessary and sufficient to alter the specificity of the MCM1 MADS box to that of MEF2, and vice versa. The role of SRF MADS-box residues which differ from those present in the other proteins was investigated by selection of functional SRF variants in yeast cells. SRF MADS-box position 1 was always a glycine in the variants, but many different sequences at the other nonconserved MADS-box residues were compatible with efficient DNA binding. We discuss potential mechanisms of DNA recognition by MADS-box proteins.
MADS盒是一种保守的序列基序，存在于一类转录因子的DNA结合结构域中，这些转录因子具有相关但不同的DNA结合特异性。我们使用嵌合蛋白和定点突变体，结合凝胶迁移率变动分析和结合位点选择分析，研究了MADS盒蛋白血清反应因子（SRF）、MCM1和MEF2A对不同序列识别的基础。删除SRF MADS盒紧邻N端的序列会使其偏好的结合位点变为MEF2A的结合位点，尽管产生的蛋白仍能微弱结合SRF特异性位点：要排他性地结合MEF2位点，还需要在MADS盒的第11至15位残基处进行进一步突变。与SRF不同，MCM1（以及MEF2A）的序列特异性完全由其MADS盒内的序列决定，仅突变SRF MADS盒的第1位残基就足以将其结合特异性改变为MCM1的结合特异性。然而，同时改变MADS盒的第1位和第11至15位才能充分改变MCM1 MADS盒对MEF2的特异性，反之亦然。通过在酵母细胞中选择功能性SRF变体，研究了与其他蛋白不同的SRF MADS盒残基的作用。在这些变体中，SRF MADS盒的第1位总是甘氨酸，但其他非保守MADS盒残基处的许多不同序列都与高效DNA结合兼容。我们讨论了MADS盒蛋白识别DNA的潜在机制。
