title: MCM1 point mutants deficient in expression of alpha-specific genes: residues important for interaction with alpha 1.
abstract: Complexes formed between MCM1 and several coregulatory proteins--alpha 1, alpha 2, and STE12--serve to govern transcription of the a- and alpha-specific gene sets in the yeast Saccharomyces cerevisiae. The N-terminal third of MCM1, MCM1(1-98), which includes a segment homologous to mammalian serum response factor, is capable of performing all of the functions necessary for cell-type-specific gene regulation, including DNA binding and interaction with coregulatory proteins. To explore the mechanisms by which MCM1(1-98) functions, we isolated point mutants that are specifically deficient in alpha-specific gene expression in vivo, anticipating that many of the mutants would be impaired for interaction with alpha 1. Indeed, in vitro DNA binding assays revealed that a substantial number of the mutants were specifically defective in the ability to bind cooperatively with alpha 1. Two other mutant classes were also found. One class, exemplified most clearly by substitutions at residues 22 and 27, exhibited a general defect in DNA binding. The second class, exemplified by substitutions at residues 33 and 41, was proficient at DNA binding and interaction with alpha 1 in vitro, suggesting that these mutants may be defective in achieving an alpha 1-mediated conformational change required for transcription activation in vivo. Most of the mutants defective for interaction with alpha 1 had substitutions within residues 69 to 81, which correspond to a region of serum response factor important for interaction with its coregulatory proteins. A subset of the mutants with changes in this region were also defective in the ability to bind with STE12 to DNA from an a-specific gene, suggesting that a common region of MCM1(1-98) mediates interaction with both alpha 1 and STE12. This region of MCM1 does not seem to constitute an independent domain of the protein, however, because some substitutions within this region affected DNA binding. Only two of the MCM1(1-98) point mutants showed significant defects in the ability to form complexes with alpha 2, suggesting that the mechanism by which MCM1 interacts with alpha 2 is distinct from that by which it interacts with alpha 1 and STE12.
authors.affiliation: Institute of Molecular Biology, University of Oregon, Eugene 97403.
authors.forename: L
authors.initials: L
authors.lastname: Bruhn
authors.forename: G F
authors.initials: GF
authors.lastname: Sprague
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D008322:Mammals; D026341:Minichromosome Maintenance 1 Protein; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000378:metabolism
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D026341:Minichromosome Maintenance 1 Protein; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D001616:beta-Galactosidase