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 me mber of the MADS-box family of transcriptional regulatory factors. To under stand 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 o f Mcm1 and examined the effects of these mutations in vivo and in vitro, Ou r results indicate which residues of Mcm1 are important for viability, tran scriptional activation, and DNA binding and bending. Substitution of residu es in Mcml which are highly conserved among the MADS-box proteins are letha l 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 alpha 2-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 conserv ed among members of the MADS-box family play important roles in contributin g 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 t hat the N-terminal extension of Mcm1 may have considerable conformational f reedom, possibly to allow binding to different DNA sites. Finally, we have identified two mutants at positions which are critical for Mcm1-mediated DN A bending that have a slow-growth phenotype. This finding is consistent wit h our earlier results, indicating that DNA bending mag have a role in Mcml function in the cell.