Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1994 Apr;14(4):2534–2544. doi: 10.1128/mcb.14.4.2534

MCM1 point mutants deficient in expression of alpha-specific genes: residues important for interaction with alpha 1.

L Bruhn 1, G F Sprague Jr 1
PMCID: PMC358621  PMID: 8139556

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.

Full text

PDF
2534

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ammerer G. Identification, purification, and cloning of a polypeptide (PRTF/GRM) that binds to mating-specific promoter elements in yeast. Genes Dev. 1990 Feb;4(2):299–312. doi: 10.1101/gad.4.2.299. [DOI] [PubMed] [Google Scholar]
  2. Bender A., Sprague G. F., Jr MAT alpha 1 protein, a yeast transcription activator, binds synergistically with a second protein to a set of cell-type-specific genes. Cell. 1987 Aug 28;50(5):681–691. doi: 10.1016/0092-8674(87)90326-6. [DOI] [PubMed] [Google Scholar]
  3. Boeke J. D., Trueheart J., Natsoulis G., Fink G. R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. doi: 10.1016/0076-6879(87)54076-9. [DOI] [PubMed] [Google Scholar]
  4. Bruhn L., Hwang-Shum J. J., Sprague G. F., Jr 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. Mol Cell Biol. 1992 Aug;12(8):3563–3572. doi: 10.1128/mcb.12.8.3563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Christ C., Tye B. K. Functional domains of the yeast transcription/replication factor MCM1. Genes Dev. 1991 May;5(5):751–763. doi: 10.1101/gad.5.5.751. [DOI] [PubMed] [Google Scholar]
  6. Dolan J. W., Fields S. Cell-type-specific transcription in yeast. Biochim Biophys Acta. 1991 Feb 16;1088(2):155–169. doi: 10.1016/0167-4781(91)90051-m. [DOI] [PubMed] [Google Scholar]
  7. Dolan J. W., Fields S. Overproduction of the yeast STE12 protein leads to constitutive transcriptional induction. Genes Dev. 1990 Apr;4(4):492–502. doi: 10.1101/gad.4.4.492. [DOI] [PubMed] [Google Scholar]
  8. Dolan J. W., Kirkman C., Fields S. The yeast STE12 protein binds to the DNA sequence mediating pheromone induction. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5703–5707. doi: 10.1073/pnas.86.15.5703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dubois E., Bercy J., Messenguy F. Characterization of two genes, ARGRI and ARGRIII required for specific regulation of arginine metabolism in yeast. Mol Gen Genet. 1987 Apr;207(1):142–148. doi: 10.1007/BF00331501. [DOI] [PubMed] [Google Scholar]
  10. Elble R., Tye B. K. Both activation and repression of a-mating-type-specific genes in yeast require transcription factor Mcm1. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10966–10970. doi: 10.1073/pnas.88.23.10966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Errede B., Ammerer G. STE12, a protein involved in cell-type-specific transcription and signal transduction in yeast, is part of protein-DNA complexes. Genes Dev. 1989 Sep;3(9):1349–1361. doi: 10.1101/gad.3.9.1349. [DOI] [PubMed] [Google Scholar]
  12. Fields S., Chaleff D. T., Sprague G. F., Jr Yeast STE7, STE11, and STE12 genes are required for expression of cell-type-specific genes. Mol Cell Biol. 1988 Feb;8(2):551–556. doi: 10.1128/mcb.8.2.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fields S., Herskowitz I. The yeast STE12 product is required for expression of two sets of cell-type specific genes. Cell. 1985 Oct;42(3):923–930. doi: 10.1016/0092-8674(85)90288-0. [DOI] [PubMed] [Google Scholar]
  14. Flessel M. C., Brake A. J., Thorner J. The MF alpha 1 gene of Saccharomyces cerevisiae: genetic mapping and mutational analysis of promoter elements. Genetics. 1989 Feb;121(2):223–236. doi: 10.1093/genetics/121.2.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grayhack E. J. The yeast alpha 1 and MCM1 proteins bind a single strand of their duplex DNA recognition site. Mol Cell Biol. 1992 Aug;12(8):3573–3582. doi: 10.1128/mcb.12.8.3573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hagen D. C., Bruhn L., Westby C. A., Sprague G. F., Jr Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1. Mol Cell Biol. 1993 Nov;13(11):6866–6875. doi: 10.1128/mcb.13.11.6866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hall M. N., Johnson A. D. Homeo domain of the yeast repressor alpha 2 is a sequence-specific DNA-binding domain but is not sufficient for repression. Science. 1987 Aug 28;237(4818):1007–1012. doi: 10.1126/science.2887035. [DOI] [PubMed] [Google Scholar]
  18. Hwang-Shum J. J., Hagen D. C., Jarvis E. E., Westby C. A., Sprague G. F., Jr Relative contributions of MCM1 and STE12 to transcriptional activation of a- and alpha-specific genes from Saccharomyces cerevisiae. Mol Gen Genet. 1991 Jun;227(2):197–204. doi: 10.1007/BF00259671. [DOI] [PubMed] [Google Scholar]
  19. Inokuchi K., Nakayama A., Hishinuma F. Identification of sequence elements that confer cell-type-specific control of MF alpha 1 expression in Saccharomyces cerevisiae. Mol Cell Biol. 1987 Sep;7(9):3185–3193. doi: 10.1128/mcb.7.9.3185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jarvis E. E., Clark K. L., Sprague G. F., Jr The yeast transcription activator PRTF, a homolog of the mammalian serum response factor, is encoded by the MCM1 gene. Genes Dev. 1989 Jul;3(7):936–945. doi: 10.1101/gad.3.7.936. [DOI] [PubMed] [Google Scholar]
  21. Jarvis E. E., Hagen D. C., Sprague G. F., Jr Identification of a DNA segment that is necessary and sufficient for alpha-specific gene control in Saccharomyces cerevisiae: implications for regulation of alpha-specific and a-specific genes. Mol Cell Biol. 1988 Jan;8(1):309–320. doi: 10.1128/mcb.8.1.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Johnson A. D., Herskowitz I. A repressor (MAT alpha 2 Product) and its operator control expression of a set of cell type specific genes in yeast. Cell. 1985 Aug;42(1):237–247. doi: 10.1016/s0092-8674(85)80119-7. [DOI] [PubMed] [Google Scholar]
  23. Keleher C. A., Goutte C., Johnson A. D. The yeast cell-type-specific repressor alpha 2 acts cooperatively with a non-cell-type-specific protein. Cell. 1988 Jun 17;53(6):927–936. doi: 10.1016/s0092-8674(88)90449-7. [DOI] [PubMed] [Google Scholar]
  24. Keleher C. A., Passmore S., Johnson A. D. Yeast repressor alpha 2 binds to its operator cooperatively with yeast protein Mcm1. Mol Cell Biol. 1989 Nov;9(11):5228–5230. doi: 10.1128/mcb.9.11.5228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kirkman-Correia C., Stroke I. L., Fields S. Functional domains of the yeast STE12 protein, a pheromone-responsive transcriptional activator. Mol Cell Biol. 1993 Jun;13(6):3765–3772. doi: 10.1128/mcb.13.6.3765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Klopotowski T., Wiater A. Synergism of aminotriazole and phosphate on the inhibition of yeast imidazole glycerol phosphate dehydratase. Arch Biochem Biophys. 1965 Dec;112(3):562–566. doi: 10.1016/0003-9861(65)90096-2. [DOI] [PubMed] [Google Scholar]
  27. Kronstad J. W., Holly J. A., MacKay V. L. A yeast operator overlaps an upstream activation site. Cell. 1987 Jul 31;50(3):369–377. doi: 10.1016/0092-8674(87)90491-0. [DOI] [PubMed] [Google Scholar]
  28. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  29. Ma H., Yanofsky M. F., Meyerowitz E. M. AGL1-AGL6, an Arabidopsis gene family with similarity to floral homeotic and transcription factor genes. Genes Dev. 1991 Mar;5(3):484–495. doi: 10.1101/gad.5.3.484. [DOI] [PubMed] [Google Scholar]
  30. Mohun T. J., Chambers A. E., Towers N., Taylor M. V. Expression of genes encoding the transcription factor SRF during early development of Xenopus laevis: identification of a CArG box-binding activity as SRF. EMBO J. 1991 Apr;10(4):933–940. doi: 10.1002/j.1460-2075.1991.tb08027.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mueller C. G., Nordheim A. A protein domain conserved between yeast MCM1 and human SRF directs ternary complex formation. EMBO J. 1991 Dec;10(13):4219–4229. doi: 10.1002/j.1460-2075.1991.tb05000.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Norman C., Runswick M., Pollock R., Treisman R. Isolation and properties of cDNA clones encoding SRF, a transcription factor that binds to the c-fos serum response element. Cell. 1988 Dec 23;55(6):989–1003. doi: 10.1016/0092-8674(88)90244-9. [DOI] [PubMed] [Google Scholar]
  33. Passmore S., Maine G. T., Elble R., Christ C., Tye B. K. Saccharomyces cerevisiae protein involved in plasmid maintenance is necessary for mating of MAT alpha cells. J Mol Biol. 1988 Dec 5;204(3):593–606. doi: 10.1016/0022-2836(88)90358-0. [DOI] [PubMed] [Google Scholar]
  34. Pollock R., Treisman R. Human SRF-related proteins: DNA-binding properties and potential regulatory targets. Genes Dev. 1991 Dec;5(12A):2327–2341. doi: 10.1101/gad.5.12a.2327. [DOI] [PubMed] [Google Scholar]
  35. Primig M., Winkler H., Ammerer G. The DNA binding and oligomerization domain of MCM1 is sufficient for its interaction with other regulatory proteins. EMBO J. 1991 Dec;10(13):4209–4218. doi: 10.1002/j.1460-2075.1991.tb04999.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rothstein R. Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol. 1991;194:281–301. doi: 10.1016/0076-6879(91)94022-5. [DOI] [PubMed] [Google Scholar]
  37. Sauer R. T., Smith D. L., Johnson A. D. Flexibility of the yeast alpha 2 repressor enables it to occupy the ends of its operator, leaving the center free. Genes Dev. 1988 Jul;2(7):807–816. doi: 10.1101/gad.2.7.807. [DOI] [PubMed] [Google Scholar]
  38. Schwarz-Sommer Z., Huijser P., Nacken W., Saedler H., Sommer H. Genetic Control of Flower Development by Homeotic Genes in Antirrhinum majus. Science. 1990 Nov 16;250(4983):931–936. doi: 10.1126/science.250.4983.931. [DOI] [PubMed] [Google Scholar]
  39. Sengupta P., Cochran B. H. The PRE and PQ box are functionally distinct yeast pheromone response elements. Mol Cell Biol. 1990 Dec;10(12):6809–6812. doi: 10.1128/mcb.10.12.6809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sharrocks A. D., Gille H., Shaw P. E. Identification of amino acids essential for DNA binding and dimerization in p67SRF: implications for a novel DNA-binding motif. Mol Cell Biol. 1993 Jan;13(1):123–132. doi: 10.1128/mcb.13.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shaw P. E. Ternary complex formation over the c-fos serum response element: p62TCF exhibits dual component specificity with contacts to DNA and an extended structure in the DNA-binding domain of p67SRF. EMBO J. 1992 Aug;11(8):3011–3019. doi: 10.1002/j.1460-2075.1992.tb05371.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sherman F. Getting started with yeast. Methods Enzymol. 1991;194:3–21. doi: 10.1016/0076-6879(91)94004-v. [DOI] [PubMed] [Google Scholar]
  43. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Song D., Dolan J. W., Yuan Y. L., Fields S. Pheromone-dependent phosphorylation of the yeast STE12 protein correlates with transcriptional activation. Genes Dev. 1991 May;5(5):741–750. doi: 10.1101/gad.5.5.741. [DOI] [PubMed] [Google Scholar]
  45. Sprague G. F., Jr Combinatorial associations of regulatory proteins and the control of cell type in yeast. Adv Genet. 1990;27:33–62. doi: 10.1016/s0065-2660(08)60023-1. [DOI] [PubMed] [Google Scholar]
  46. Stevenson B. J., Rhodes N., Errede B., Sprague G. F., Jr Constitutive mutants of the protein kinase STE11 activate the yeast pheromone response pathway in the absence of the G protein. Genes Dev. 1992 Jul;6(7):1293–1304. doi: 10.1101/gad.6.7.1293. [DOI] [PubMed] [Google Scholar]
  47. Struhl K., Davis R. W. Production of a functional eukaryotic enzyme in Escherichia coli: cloning and expression of the yeast structural gene for imidazole-glycerolphosphate dehydratase (his3). Proc Natl Acad Sci U S A. 1977 Dec;74(12):5255–5259. doi: 10.1073/pnas.74.12.5255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tan S., Ammerer G., Richmond T. J. Interactions of purified transcription factors: binding of yeast MAT alpha 1 and PRTF to cell type-specific, upstream activating sequences. EMBO J. 1988 Dec 20;7(13):4255–4264. doi: 10.1002/j.1460-2075.1988.tb03323.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tan S., Richmond T. J. DNA binding-induced conformational change of the yeast transcriptional activator PRTF. Cell. 1990 Jul 27;62(2):367–377. doi: 10.1016/0092-8674(90)90373-m. [DOI] [PubMed] [Google Scholar]
  50. Vershon A. K., Johnson A. D. A short, disordered protein region mediates interactions between the homeodomain of the yeast alpha 2 protein and the MCM1 protein. Cell. 1993 Jan 15;72(1):105–112. doi: 10.1016/0092-8674(93)90054-t. [DOI] [PubMed] [Google Scholar]
  51. Yuan Y. O., Stroke I. L., Fields S. Coupling of cell identity to signal response in yeast: interaction between the alpha 1 and STE12 proteins. Genes Dev. 1993 Aug;7(8):1584–1597. doi: 10.1101/gad.7.8.1584. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES