MCM1 Protein Research Articles

A short, disordered protein region mediates interactions between the homeodomain of the yeast α2 protein and the MCM1 protein

Homeodomains are folded into a characteristic three-dimensional structure capable of recognizing DNA in a sequence-specific manner. We show that correct target site selection by the yeast α2 protein requires, as well as its homeodomain, an adjacent short and apparently unstructured region of the protein. This flexible homeodomain extension is responsible for specifying an interaction with a second regulatory protein, MCM1, which permits the cooperative binding of the two proteins to an operator. Two additional experiments suggest that this extension-homeodomain arrangement is likely to have some generality. First, when the extension of α2 is grafted onto the Drosophila engrailed homeodomain, it yields a protein with the DNA binding specificity of engrailed and the ability to bind cooperatively to DNA with MCM1. Second, the α2 extension specifies interaction not only with the yeast MCM1 protein, but also with the related human protein SRF.

SPT13 (GAL11) of Saccharomyces cerevisiae negatively regulates activity of the MCM1 transcription factor in Ty1 elements.

The Ty transposable elements of Saccharomyces cerevisiae consist of a single large transcription unit whose expression is controlled by a combination of upstream and downstream regulatory sequences. Errede (B. Errede, Mol. Cell. Biol. 13:57-62, 1993) has shown that among the downstream control sequences is a binding site for the transcription factor, MCM1. A small restriction fragment containing the Ty1 MCM1-binding site exhibits very weak activation of heterologous gene expression. The absence of SPT13 (GAL11) causes a dramatic increase in activity directed by these sequences. This effect is mediated through the MCM1-binding site itself. MCM1 mRNA and protein levels, as well as its affinity for its binding site, are unchanged in the absence of SPT13. Our results suggest that SPT13 has a role in the negative control of MCM1 activity that is likely to be posttranslational. A role for SPT13 in the negative regulation of the activity of the Ty1 MCM1-binding site is consistent with our previous proposal that spt13-mediated suppression of Ty insertion mutations could be attributed to the loss of negative regulation of genes adjacent to Ty elements.

SPT13 (GAL11) of Saccharomyces cerevisiae negatively regulates activity of the MCM1 transcription factor in Ty1 elements.

The Ty transposable elements of Saccharomyces cerevisiae consist of a single large transcription unit whose expression is controlled by a combination of upstream and downstream regulatory sequences. Errede (B. Errede, Mol. Cell. Biol. 13:57-62, 1993) has shown that among the downstream control sequences is a binding site for the transcription factor, MCM1. A small restriction fragment containing the Ty1 MCM1-binding site exhibits very weak activation of heterologous gene expression. The absence of SPT13 (GAL11) causes a dramatic increase in activity directed by these sequences. This effect is mediated through the MCM1-binding site itself. MCM1 mRNA and protein levels, as well as its affinity for its binding site, are unchanged in the absence of SPT13. Our results suggest that SPT13 has a role in the negative control of MCM1 activity that is likely to be posttranslational. A role for SPT13 in the negative regulation of the activity of the Ty1 MCM1-binding site is consistent with our previous proposal that spt13-mediated suppression of Ty insertion mutations could be attributed to the loss of negative regulation of genes adjacent to Ty elements.

The Yeast a1 and MCM1 Proteins Bind a Single Strand of Their Duplex DNA Recognition Site

The yeast cell type regulator alpha 1 cooperates with a constitutive factor, MCM1 protein, to recognize the promoter and activate transcription of several alpha-specific genes. I show here that the alpha 1 and MCM1 proteins bind specifically to one of the two strands of their recognition sequence. This single-strand-binding activity shares several characteristics with the duplex-binding properties of these proteins: (i) the MCM1 protein binds alone to single-stranded and duplex sequences of both the alpha-specific (P'Q) and a-specific (P) binding sites; (ii) the alpha 1 protein requires both the MCM1 protein and the Q sequence to bind either single-stranded or duplex DNA; (iii) the alpha 1 protein stimulates binding of the MCM1 protein to both single-stranded and duplex DNAs; and (iv) the affinities of the proteins for single-stranded and duplex DNAs are comparable.

The yeast alpha 1 and MCM1 proteins bind a single strand of their duplex DNA recognition site.

The yeast cell type regulator alpha 1 cooperates with a constitutive factor, MCM1 protein, to recognize the promoter and activate transcription of several alpha-specific genes. I show here that the alpha 1 and MCM1 proteins bind specifically to one of the two strands of their recognition sequence. This single-strand-binding activity shares several characteristics with the duplex-binding properties of these proteins: (i) the MCM1 protein binds alone to single-stranded and duplex sequences of both the alpha-specific (P'Q) and a-specific (P) binding sites; (ii) the alpha 1 protein requires both the MCM1 protein and the Q sequence to bind either single-stranded or duplex DNA; (iii) the alpha 1 protein stimulates binding of the MCM1 protein to both single-stranded and duplex DNAs; and (iv) the affinities of the proteins for single-stranded and duplex DNAs are comparable.

A molecular mechanism for combinatorial control in yeast: MCM1 protein sets the spacing and orientation of the homeodomains of an α2 dimer: D.L. SMITH AND A.D. JOHNSON Cell 68, 133–142

A molecular mechanism for combinatorial control in yeast: MCM1 protein sets the spacing and orientation of the homeodomains of an α2 dimer: D.L. SMITH AND A.D. JOHNSON Cell 68, 133–142

A molecular mechanism for combinatorial control in yeast: MCM1 protein sets the spacing and orientation of the homeodomains of an α2 dimer

DNA recognition sequences for dimeric proteins typically contain two types of information. The first is the DNA sequence of each half-site, and the second is the arrangement of these half-sites. We show that dimers of the yeast homeodomain protein α2, although able to read the first type of information, lack the ability to assess the second type. Rather, α2 dimers bind with equal affinity to artifical operators in which the two half-sites are arrayed as inverted repeats, as direct repeats, or as everted (inside-out) repeats. We show that a second protein — MCM1 — sets the exact spacing and orientation of the homeodomains in the α2 dimer so that they accommodate only the geometry of the naturally occurring operators. These experiments show directly how the target specificity of a homeodomain protein is raised by an auxiliary protein, allowing it to distinguish the biologically correct operators from closely related sequences in the cell.

A molecular mechanism for combinatorial control in yeast: MCM1 protein sets the spacing and orientation of the homeodomains of an α2 dimer D.L. SMITH AND A.D. JOHNSON Cell 68, 133–142

A molecular mechanism for combinatorial control in yeast: MCM1 protein sets the spacing and orientation of the homeodomains of an α2 dimer D.L. SMITH AND A.D. JOHNSON Cell 68, 133–142

Both activation and repression of a-mating-type-specific genes in yeast require transcription factor Mcm1.

Mcm1 is a yeast transcription factor with homologs throughout the metazoa. MCM1 was first identified as a gene involved in maintenance of artificial minichromosomes in yeast. More recently Mcm1 has been shown to serve as a transcriptional regulator of mating-type-specific genes. Biochemical data suggest that Mcm1 coactivates alpha-specific genes and corepresses a-specific genes by binding to a 10-base-pair dyad symmetry element in their upstream regions. We reported previously that an mcm1 point mutation reduced activation of alpha-specific genes but had little effect on the expression of a-specific genes. We now show that another mcm1 allele, which depletes the Mcm1 protein, affects both activation and repression of a-specific genes. The mutant strain remains capable of high levels of pheromone induction of a-specific genes, although with retarded kinetics. Mcm1 joins an increasing number of transcription factors involved in both positive and negative regulation of gene expression.

The DNA binding and oligomerization domain of MCM1 is sufficient for its interaction with other regulatory proteins.

The MCM1 gene encodes an essential DNA binding protein that, in cooperation with the transactivators alpha 1 and STE12 and the repressor alpha 2, confers mating specificity to haploid yeast cells. We show that the amino-terminal third of the MCM1 protein is sufficient for the physical interaction with these factors. A strain expressing just 98 amino acids encompassing the oligomerization and DNA binding domains of MCM1 is viable and mating competent. This motif exhibits considerable similarity to a domain of the mammalian transcription factor SRF. A 98 amino acid hybrid gene coding for the MCM1 DNA binding domain and SRF dimerization domain is sufficient for viability but not for the expression of mating type specific genes. In vitro binding studies suggest that a region of approximately 50 amino acids of MCM1 is essential for providing contacts with alpha 1, alpha 2 and STE12.

The PRE and PQ Box Are Functionally Distinct Yeast Pheromone Response Elements

Saccharomyces cerevisiae mating pheromones function by binding to cell surface receptors and activating signal transduction processes which regulate gene expression. In this report, we have analyzed the minimum sequence requirements for conferring both a and alpha mating pheromone inducibilities onto a heterologous promoter. Here we show that the repetitive pheromone response element (PRE) which binds to STE12 protein is sufficient to confer pheromone responsiveness only when present in multiple copies. Moreover, by itself, it is preferentially responsive to alpha factor in a cells. In contrast, a single copy of the PQ box of the STE3 upstream activation sequence (UAS) is sufficient to confer a-factor responsiveness in alpha cells. The PQ box binds both MCM1 and MAT alpha 1 in a cooperative manner, and neither the P nor Q site alone is sufficient to confer a-factor responsiveness. In a cells, however, even multiple copies of the PQ box fail to confer alpha-factor responsiveness. Therefore, the PRE and the PQ box are functionally distinct pheromone-responsive elements with opposite cell type specificities. Moreover, these results indicate that the MCM1 protein functions in a signal transduction pathway in a manner analogous to that of its mammalian homolog, the serum response factor, which regulates the expression of the c-fos proto-oncogene in mammals.

The PRE and PQ box are functionally distinct yeast pheromone response elements.

Saccharomyces cerevisiae mating pheromones function by binding to cell surface receptors and activating signal transduction processes which regulate gene expression. In this report, we have analyzed the minimum sequence requirements for conferring both a and alpha mating pheromone inducibilities onto a heterologous promoter. Here we show that the repetitive pheromone response element (PRE) which binds to STE12 protein is sufficient to confer pheromone responsiveness only when present in multiple copies. Moreover, by itself, it is preferentially responsive to alpha factor in a cells. In contrast, a single copy of the PQ box of the STE3 upstream activation sequence (UAS) is sufficient to confer a-factor responsiveness in alpha cells. The PQ box binds both MCM1 and MAT alpha 1 in a cooperative manner, and neither the P nor Q site alone is sufficient to confer a-factor responsiveness. In a cells, however, even multiple copies of the PQ box fail to confer alpha-factor responsiveness. Therefore, the PRE and the PQ box are functionally distinct pheromone-responsive elements with opposite cell type specificities. Moreover, these results indicate that the MCM1 protein functions in a signal transduction pathway in a manner analogous to that of its mammalian homolog, the serum response factor, which regulates the expression of the c-fos proto-oncogene in mammals.

Functional analysis of ARGRI and ARGRIII regulatory proteins involved in the regulation of arginine metabolism inSaccharomyces cerevisiae

We present here a functional analysis of ARGRI and ARGRIII regulatory proteins which are involved together with ARGRII in specific regulation of arginine anabolic and catabolic pathways. Unlike ARGRII, ARGRI and ARGRIII have no transcriptional activation capacity. The first 60 amino acids of ARGRI (out of 177) are dispensable for its activity. The functional domain of the protein is located in the region of homology with MCM1 and SRF proteins. ARGRIII contains in its C-terminal portion a stretch of 17 aspartate residues which are indispensable for arginine regulation. Gene disruption of the ARGRIII gene impairs the growth of the mutant on rich medium, showing that ARGRIII has a pleiotropic role in the cell.

The yeast transcription activator PRTF, a homolog of the mammalian serum response factor, is encoded by the MCM1 gene.

Two proteins, alpha 1 and pheromone/receptor transcription factor (PRTF), bind cooperatively to the upstream activation sequences (UAS) of yeast alpha-specific genes and thereby activate their transcription. In these protein-DNA complexes, the PRTF moiety interacts with a degenerate dyad symmetric sequence, the P box. PRTF contributes also to the regulation of a second set of cell-type-specific genes, the a-specific genes. We used two in vitro assays to show that PRTF is encoded, at least in part, by the MCM1 gene. In one assay, truncated MCM1 proteins encoded by deletion derivatives of the MCM1 gene formed protein-DNA complexes of novel mobility, demonstrated that MCM1 can bind to the P-box-containing DNA. Second, antibodies raised to a synthetic MCM1 polypeptide retard the migration of PRTF-DNA complexes in gel mobility shift assays. This result indicates that PRTF, defined as an activity that binds cooperatively with alpha 1 to alpha-specific UAS elements, shares an epitope with MCM1. In addition, we show that MCM1 deletions that remove the carboxy-terminal 129 codons of 286 total codons encode truncated MCM1 molecules that are competent to activate transcription in vivo, indicating that the carboxy-terminal residues are not required for this process.

Saccharomyces cerevisiae protein involved in plasmid maintenance is necessary for mating of MATα cells

We previously reported the isolation of yeast mutants that seem to affect the function of certain autonomously replicating sequences ( ARSs). These mutants are known as mcm for their defect in the maintenance of minichromosomes. We have now characterized in more detail one ARS-specific mutation, mcm1-1. This Mcm1 mutant has a second phenotype; MATα mcm1-1 strains are sterile. MCM1 is non-allelic to other known α-specific sterile mutations and, unlike most genes required for mating, it is essential for growth. The α-specific sterile phenotype of the mcm1-1 mutant is manifested by its failure to produce a normal amount of the mating pheromone, α-factor. In addition, transcripts of the MFα1 and STE3 genes, which encode the α-factor precursor and the a-factor receptor, respectively, are greatly reduced in this mutant. These and other properties of the mcm1-1 mutant suggest that the MCM1 protein may act as a transcriptional activator of α-specific genes. We have cloned, mapped and sequenced the wild-type and mutant alleles of MCM1, which is located on the right arm of chromosome XIII near LYS7. The MCM1 gene product is a protein of 286 amino acid residues and contains an unusual region in which 19 out of 20 residues are either aspartic or glutamic acid, followed by a series of glutamine tracts. MCM1 has striking homology to ARG80, a regulatory gene of the arginine metabolic pathway located about 700 base-pairs upstream from MCM1. A substitution of leucine for proline at amino acid position 97, immediately preceding the polyanionic region, was shown to be responsible for both the α-specific sterile and minichromosome-maintenance defective phenotypes of the mcm1-1 mutant.