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 Mcm1 in vivo and in vitro, we have introduced an extensive series of base pair substitutions into an Mcm1 operator site and examined their effects on Mcm1-mediated transcriptional regulation and DNA-binding affinity. Our results show that Mcm1 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 Mcm1 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)6GG 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 Mcm1 is sequence dependent and that the base-specific requirements for bending differ between Mcm1 and SRF. Interestingly, although these substitutions have a large effect on DNA bending and transcriptional activation by Mcm1, 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.
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Waksman Institute of Microbiology, Rutgers University, Piscataway, New Jersey 08855-0759, USA.
Zhong
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Vershon
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D026341:Minichromosome Maintenance 1 Protein; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D026341:Minichromosome Maintenance 1 Protein; D014157:Transcription Factors