The N-terminal 96 residues of MCM1, a regulator of cell type-specific genes in Saccharomyces cerevisiae, are sufficient for DNA binding, transcription activation, and interaction with alpha 1.
MCM1 performs several functions necessary for its role in regulating cell type-specific gene expression in the yeast Saccharomyces cerevisiae: DNA binding, transcription activation, and interaction with coregulatory proteins such as alpha 1. We analyzed a set of MCM1 deletion derivatives using in vivo reporter gene assays and in vitro DNA-binding studies to determine which regions of MCM1 are important for its various activities. We also analyzed a set of LexA-MCM1 hybrids to examine the ability of different segments of MCM1 to activate transcription independent of MCM1's DNA-binding function. The first third of MCM1 [MCM1(1-96)], which includes an 80-residue segment homologous to the mammalian serum response factor, was sufficient for high-affinity DNA binding, for activation of reporter gene expression, and for interaction with alpha 1 in vitro and in vivo. However, the ability of MCM1(1-96) to activate transcription and to interact with alpha 1 was somewhat reduced compared with wild-type MCM1 [MCM1(1-286)]. Optimal interaction with alpha 1 required residues 99 to 117, in which 18 of 19 amino acids are acidic in character. Optimal transcription activation required a segment from residues 188 to 286, in which 50% of the amino acids are glutamine. Deletion of this segment from MCM1 reduced expression of reporter genes by about twofold. Moreover, LexA-MCM1 hybrids containing this segment were able to activate expression of reporter genes that rely on LexA binding sites as potential upstream activation sequences. Thus, glutamine-rich regions may contribute to the activation function of yeast transcription activators, as has been suggested for glutamine-rich mammalian proteins such as Sp1.
Bruhn
L
L
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Hwang-Shum
J J
JJ
Sprague
G F
GF
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002872:Chromosome Deletion; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D007700:Kinetics; D000072235:Mating Factor; D026341:Minichromosome Maintenance 1 Protein; D010455:Peptides / Q000378:metabolism*; D010675:Pheromones / Q000378:metabolism; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D012697:Serine Endopeptidases; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D000072235:Mating Factor; D026341:Minichromosome Maintenance 1 Protein; D010455:Peptides; D010675:Pheromones; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D001616:beta-Galactosidase; D000099160:LexA Repressor Protein