Scanning Mutagenesis of Mcm1: Residues Required for DNA Binding, DNA Bending, and Transcriptional Activation by a MADS-Box Protein

MCM1 is an essential gene in the yeast Saccharomyces cerevisiae and is a member of the MADS-box family of transcriptional regulatory factors. To understand the nature of the protein-DNA interactions of this class of proteins, we have made a series of alanine substitutions in the DNA-binding domain of Mcm1 and examined the effects of these mutations in vivo and in vitro. Our results indicate which residues of Mcm1 are important for viability, transcriptional activation, and DNA binding and bending. Substitution of residues in Mcm1 which are highly conserved among the MADS-box proteins are lethal to the cell and abolish DNA binding in vitro. These positions have almost identical interactions with DNA in both the serum response factor-DNA and α2-Mcm1-DNA crystal structures, suggesting that these residues make up a conserved core of protein-DNA interactions responsible for docking MADS-box proteins to DNA. Substitution of residues which are not as well conserved among members of the MADS-box family play important roles in contributing to the specificity of DNA binding. These results suggest a general model of how MADS-box proteins recognize and bind DNA. We also provide evidence that the N-terminal extension of Mcm1 may have considerable conformational freedom, possibly to allow binding to different DNA sites. Finally, we have identified two mutants at positions which are critical for Mcm1-mediated DNA bending that have a slow-growth phenotype. This finding is consistent with our earlier results, indicating that DNA bending may have a role in Mcm1 function in the cell.

MCM1是酿酒酵母（Saccharomyces cerevisiae）的必需基因，隶属于转录调控因子的MADS盒（MADS-box）家族。为阐明该类蛋白质的蛋白质-DNA相互作用本质，我们对Mcm1的DNA结合结构域开展了一系列丙氨酸替换突变，并在体内与体外环境中检测了这些突变的效应。本研究结果明确了Mcm1中哪些残基对细胞存活、转录激活以及DNA结合与弯曲过程具有关键作用。在MADS盒家族蛋白质中高度保守的Mcm1残基发生替换后，会导致细胞死亡，并在体外完全丧失DNA结合能力。在血清应答因子（serum response factor）-DNA与α2-Mcm1-DNA的晶体结构中，这些残基位点与DNA的相互作用模式几乎完全一致，这表明这些残基构成了蛋白质-DNA相互作用的保守核心，负责将MADS盒家族蛋白质锚定至DNA分子上。MADS盒家族成员间保守性较低的残基发生替换，则对DNA结合的特异性发挥重要调控作用。上述研究结果提出了MADS盒家族蛋白质识别并结合DNA的通用模型。本研究同时提供证据表明，Mcm1的N端延伸区域可能具有显著的构象自由度，这或许使其能够结合不同的DNA位点。最后，本研究鉴定出两个位于Mcm1介导的DNA弯曲关键位点的突变体，它们呈现出生长缓慢的表型。这一发现与我们此前的研究结果相符，表明DNA弯曲过程可能在细胞内Mcm1的功能发挥中具有重要作用。