DNA-binding specificity of Mcm1: Operator mutations that alter DNA-bending and transcriptional activities by a MADS box protein

The yeast Mcm1 protein is a member of the MADS box family of transcriptional regulatory factors, a class of DNA-binding proteins found in such diverse organisms as yeast, plants, flies, and humans. To explore the protein-DNA interactions of Mcml in vivo and in vitro, we have introduced an extensive series of base pair substitutions into an Mcml operator site and examined their effects on Mcm1-mediated transcriptional regulation and DNA-binding affinity. Our results show that Mcml uses a mechanism to contact the DNA that has some significant differences from the one used by the human serum response factor (SRF), a closely related MADS box protein in which the three-dimensional structure has been determined. One major difference is that 5-bromouracil-mediated photo-cross-linking experiments indicate that Mcml is in close proximity to functional groups in the major groove at the center of the recognition site whereas the SRF protein did not exhibit this characteristic, A more significant difference is that mutations at a position outside of the conserved CC(A/T)(6)GG site significantly reduce Mcm1-dependent DNA bending, while these substitutions have no effect on DNA bending by SRF. This result shows that the DNA bending by Mcml is sequence dependent and that the base-specific requirements for bending differ between Mcml and SRF, Interestingly, although these substitutions have a large effect on DNA bending and transcriptional activation by Mcml, they have a relatively small effect on the DNA-binding affinity of the protein. This result suggests that the degree of DNA bending is important for transcriptional activation by Mcm1.