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
. 1997 Apr;17(4):1881–1889. doi: 10.1128/mcb.17.4.1881

DNA-binding specificity of Mcm1: operator mutations that alter DNA-bending and transcriptional activities by a MADS box protein.

T B Acton 1, H Zhong 1, A K Vershon 1
PMCID: PMC232035  PMID: 9121436

Abstract

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.

Full Text

The Full Text of this article is available as a PDF (767.2 KB).

Selected References

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

  1. Affolter M., Montagne J., Walldorf U., Groppe J., Kloter U., LaRosa M., Gehring W. J. The Drosophila SRF homolog is expressed in a subset of tracheal cells and maps within a genomic region required for tracheal development. Development. 1994 Apr;120(4):743–753. doi: 10.1242/dev.120.4.743. [DOI] [PubMed] [Google Scholar]
  2. Allen T. D., Wick K. L., Matthews K. S. Identification of amino acids in lac repressor protein cross-linked to operator DNA specifically substituted with bromodeoxyuridine. J Biol Chem. 1991 Apr 5;266(10):6113–6119. [PubMed] [Google Scholar]
  3. 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]
  4. Blatter E. E., Ebright Y. W., Ebright R. H. Identification of an amino acid-base contact in the GCN4-DNA complex by bromouracil-mediated photocrosslinking. Nature. 1992 Oct 15;359(6396):650–652. doi: 10.1038/359650a0. [DOI] [PubMed] [Google Scholar]
  5. Bruhn L., Sprague G. F., Jr MCM1 point mutants deficient in expression of alpha-specific genes: residues important for interaction with alpha 1. Mol Cell Biol. 1994 Apr;14(4):2534–2544. doi: 10.1128/mcb.14.4.2534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carey J. Gel retardation. Methods Enzymol. 1991;208:103–117. doi: 10.1016/0076-6879(91)08010-f. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Dong Q., Blatter E. E., Ebright Y. W., Bister K., Ebright R. H. Identification of amino acid-base contacts in the Myc-DNA complex by site-specific bromouracil mediated photocrosslinking. EMBO J. 1994 Jan 1;13(1):200–204. doi: 10.1002/j.1460-2075.1994.tb06249.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dubois E., Bercy J., Descamps F., Messenguy F. Characterization of two new genes essential for vegetative growth in Saccharomyces cerevisiae: nucleotide sequence determination and chromosome mapping. Gene. 1987;55(2-3):265–275. doi: 10.1016/0378-1119(87)90286-1. [DOI] [PubMed] [Google Scholar]
  10. Guarente L., Hoar E. Upstream activation sites of the CYC1 gene of Saccharomyces cerevisiae are active when inverted but not when placed downstream of the "TATA box". Proc Natl Acad Sci U S A. 1984 Dec;81(24):7860–7864. doi: 10.1073/pnas.81.24.7860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gustafson T. A., Taylor A., Kedes L. DNA bending is induced by a transcription factor that interacts with the human c-FOS and alpha-actin promoters. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2162–2166. doi: 10.1073/pnas.86.7.2162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Herskowitz I. A regulatory hierarchy for cell specialization in yeast. Nature. 1989 Dec 14;342(6251):749–757. doi: 10.1038/342749a0. [DOI] [PubMed] [Google Scholar]
  13. Hill C. S., Marais R., John S., Wynne J., Dalton S., Treisman R. Functional analysis of a growth factor-responsive transcription factor complex. Cell. 1993 Apr 23;73(2):395–406. doi: 10.1016/0092-8674(93)90238-l. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Lilly B., Galewsky S., Firulli A. B., Schulz R. A., Olson E. N. D-MEF2: a MADS box transcription factor expressed in differentiating mesoderm and muscle cell lineages during Drosophila embryogenesis. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5662–5666. doi: 10.1073/pnas.91.12.5662. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lydall D., Ammerer G., Nasmyth K. A new role for MCM1 in yeast: cell cycle regulation of SW15 transcription. Genes Dev. 1991 Dec;5(12B):2405–2419. doi: 10.1101/gad.5.12b.2405. [DOI] [PubMed] [Google Scholar]
  18. Maher M., Cong F., Kindelberger D., Nasmyth K., Dalton S. Cell cycle-regulated transcription of the CLB2 gene is dependent on Mcm1 and a ternary complex factor. Mol Cell Biol. 1995 Jun;15(6):3129–3137. doi: 10.1128/mcb.15.6.3129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mead J., Zhong H., Acton T. B., Vershon A. K. The yeast alpha2 and Mcm1 proteins interact through a region similar to a motif found in homeodomain proteins of higher eukaryotes. Mol Cell Biol. 1996 May;16(5):2135–2143. doi: 10.1128/mcb.16.5.2135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Natesan S., Gilman M. Z. DNA bending and orientation-dependent function of YY1 in the c-fos promoter. Genes Dev. 1993 Dec;7(12B):2497–2509. doi: 10.1101/gad.7.12b.2497. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Nurrish S. J., Treisman R. DNA binding specificity determinants in MADS-box transcription factors. Mol Cell Biol. 1995 Aug;15(8):4076–4085. doi: 10.1128/mcb.15.8.4076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pabo C. O., Sauer R. T. Transcription factors: structural families and principles of DNA recognition. Annu Rev Biochem. 1992;61:1053–1095. doi: 10.1146/annurev.bi.61.070192.005201. [DOI] [PubMed] [Google Scholar]
  25. Parekh B. S., Hatfield G. W. Transcriptional activation by protein-induced DNA bending: evidence for a DNA structural transmission model. Proc Natl Acad Sci U S A. 1996 Feb 6;93(3):1173–1177. doi: 10.1073/pnas.93.3.1173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Passmore S., Elble R., Tye B. K. A protein involved in minichromosome maintenance in yeast binds a transcriptional enhancer conserved in eukaryotes. Genes Dev. 1989 Jul;3(7):921–935. doi: 10.1101/gad.3.7.921. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Pellegrini L., Tan S., Richmond T. J. Structure of serum response factor core bound to DNA. Nature. 1995 Aug 10;376(6540):490–498. doi: 10.1038/376490a0. [DOI] [PubMed] [Google Scholar]
  29. Pollock R., Treisman R. A sensitive method for the determination of protein-DNA binding specificities. Nucleic Acids Res. 1990 Nov 11;18(21):6197–6204. doi: 10.1093/nar/18.21.6197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Riechmann J. L., Wang M., Meyerowitz E. M. DNA-binding properties of Arabidopsis MADS domain homeotic proteins APETALA1, APETALA3, PISTILLATA and AGAMOUS. Nucleic Acids Res. 1996 Aug 15;24(16):3134–3141. doi: 10.1093/nar/24.16.3134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Sharrocks A. D., Shore P. DNA bending in the ternary nucleoprotein complex at the c-fos promoter. Nucleic Acids Res. 1995 Jul 11;23(13):2442–2449. doi: 10.1093/nar/23.13.2442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shaw P. E., Schröter H., Nordheim A. The ability of a ternary complex to form over the serum response element correlates with serum inducibility of the human c-fos promoter. Cell. 1989 Feb 24;56(4):563–572. doi: 10.1016/0092-8674(89)90579-5. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Siliciano P. G., Tatchell K. Transcription and regulatory signals at the mating type locus in yeast. Cell. 1984 Jul;37(3):969–978. doi: 10.1016/0092-8674(84)90431-8. [DOI] [PubMed] [Google Scholar]
  36. Smith D. L., Desai A. B., Johnson A. D. DNA bending by the a1 and alpha 2 homeodomain proteins from yeast. Nucleic Acids Res. 1995 Apr 11;23(7):1239–1243. doi: 10.1093/nar/23.7.1239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Smith D. L., Johnson A. D. Operator-constitutive mutations in a DNA sequence recognized by a yeast homeodomain. EMBO J. 1994 May 15;13(10):2378–2387. doi: 10.1002/j.1460-2075.1994.tb06521.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sommer H., Beltrán J. P., Huijser P., Pape H., Lönnig W. E., Saedler H., Schwarz-Sommer Z. Deficiens, a homeotic gene involved in the control of flower morphogenesis in Antirrhinum majus: the protein shows homology to transcription factors. EMBO J. 1990 Mar;9(3):605–613. doi: 10.1002/j.1460-2075.1990.tb08152.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. Thompson J. F., Landy A. Empirical estimation of protein-induced DNA bending angles: applications to lambda site-specific recombination complexes. Nucleic Acids Res. 1988 Oct 25;16(20):9687–9705. doi: 10.1093/nar/16.20.9687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Treisman R. Identification and purification of a polypeptide that binds to the c-fos serum response element. EMBO J. 1987 Sep;6(9):2711–2717. doi: 10.1002/j.1460-2075.1987.tb02564.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Treisman R. Identification of a protein-binding site that mediates transcriptional response of the c-fos gene to serum factors. Cell. 1986 Aug 15;46(4):567–574. doi: 10.1016/0092-8674(86)90882-2. [DOI] [PubMed] [Google Scholar]
  44. Treisman R. Journey to the surface of the cell: Fos regulation and the SRE. EMBO J. 1995 Oct 16;14(20):4905–4913. doi: 10.1002/j.1460-2075.1995.tb00173.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tröbner W., Ramirez L., Motte P., Hue I., Huijser P., Lönnig W. E., Saedler H., Sommer H., Schwarz-Sommer Z. GLOBOSA: a homeotic gene which interacts with DEFICIENS in the control of Antirrhinum floral organogenesis. EMBO J. 1992 Dec;11(13):4693–4704. doi: 10.1002/j.1460-2075.1992.tb05574.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. Willis M. C., Hicke B. J., Uhlenbeck O. C., Cech T. R., Koch T. H. Photocrosslinking of 5-iodouracil-substituted RNA and DNA to proteins. Science. 1993 Nov 19;262(5137):1255–1257. doi: 10.1126/science.7694369. [DOI] [PubMed] [Google Scholar]
  48. Wolberger C., Vershon A. K., Liu B., Johnson A. D., Pabo C. O. Crystal structure of a MAT alpha 2 homeodomain-operator complex suggests a general model for homeodomain-DNA interactions. Cell. 1991 Nov 1;67(3):517–528. doi: 10.1016/0092-8674(91)90526-5. [DOI] [PubMed] [Google Scholar]
  49. Wu H. M., Crothers D. M. The locus of sequence-directed and protein-induced DNA bending. Nature. 1984 Apr 5;308(5959):509–513. doi: 10.1038/308509a0. [DOI] [PubMed] [Google Scholar]
  50. Wynne J., Treisman R. SRF and MCM1 have related but distinct DNA binding specificities. Nucleic Acids Res. 1992 Jul 11;20(13):3297–3303. doi: 10.1093/nar/20.13.3297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Yanofsky M. F., Ma H., Bowman J. L., Drews G. N., Feldmann K. A., Meyerowitz E. M. The protein encoded by the Arabidopsis homeotic gene agamous resembles transcription factors. Nature. 1990 Jul 5;346(6279):35–39. doi: 10.1038/346035a0. [DOI] [PubMed] [Google Scholar]
  52. Yu G., Deschenes R. J., Fassler J. S. The essential transcription factor, Mcm1, is a downstream target of Sln1, a yeast "two-component" regulator. J Biol Chem. 1995 Apr 14;270(15):8739–8743. doi: 10.1074/jbc.270.15.8739. [DOI] [PubMed] [Google Scholar]

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

RESOURCES