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. 1997 Oct;147(2):493–505. doi: 10.1093/genetics/147.2.493

Specialized Rap1p/Gcr1p Transcriptional Activation through Gcr1p DNA Contacts Requires Gcr2p, as Does Hyperphosphorylation of Gcr1p

X Zeng 1, S J Deminoff 1, G M Santangelo 1
PMCID: PMC1208173  PMID: 9335588

Abstract

The multifunctional regulatory factor Rap1p of Saccharomyces cerevisiae accomplishes one of its tasks, transcriptional activation, by complexing with Gcr1p. An unusual feature of this heteromeric complex is its apparent capacity to contact simultaneously two adjacent DNA elements (UAS(RPG) and the CT box, bound specifically by Rap1p and Gcr1p, respectively). The complex can activate transcription through isolated UAS(RPG) but not CT elements. In promoters that contain both DNA signals its activity is enhanced, provided the helical spacing between the two elements is appropriate; this suggests that at least transient DNA loop formation is involved. We show here that this CT box-dependent augmentation of Rap1p/Gcr1p activation requires the presence of a third protein Gcr2p; the Gcr2(-) growth defect appears to result from a genome-wide loss of the CT box effect. Interestingly, a hyperphosphorylated form of Gcr1p disappears in Δgcr2 cells but reappears if they harbor a doubly point-mutated GCR1 allele that bypasses the Gcr2(-) growth defect. Gcr2p therefore appears to induce a conformation change in Gcr1p and/or stimulate its hyperphosphorylation; one or both of these effects can be mimicked in the absence of GCR2 by mutation of GCR1. This improved view of Rap1p/Gcr1p/Gcr2p function reveals a new aspect of eukaryotic gene regulation: modification of an upstream activator, accompanied by at least transient DNA loop formation, mediates its improved capacity to activate transcription.

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Selected References

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  1. Bitter G. A., Chang K. K., Egan K. M. A multi-component upstream activation sequence of the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene promoter. Mol Gen Genet. 1991 Dec;231(1):22–32. doi: 10.1007/BF00293817. [DOI] [PubMed] [Google Scholar]
  2. Boyle W. J., van der Geer P., Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. doi: 10.1016/0076-6879(91)01013-r. [DOI] [PubMed] [Google Scholar]
  3. Buchman A. R., Lue N. F., Kornberg R. D. Connections between transcriptional activators, silencers, and telomeres as revealed by functional analysis of a yeast DNA-binding protein. Mol Cell Biol. 1988 Dec;8(12):5086–5099. doi: 10.1128/mcb.8.12.5086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  5. Deminoff S. J., Tornow J., Santangelo G. M. Unigenic evolution: a novel genetic method localizes a putative leucine zipper that mediates dimerization of the Saccharomyces cerevisiae regulator Gcr1p. Genetics. 1995 Dec;141(4):1263–1274. doi: 10.1093/genetics/141.4.1263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Drazinic C. M., Smerage J. B., López M. C., Baker H. V. Activation mechanism of the multifunctional transcription factor repressor-activator protein 1 (Rap1p). Mol Cell Biol. 1996 Jun;16(6):3187–3196. doi: 10.1128/mcb.16.6.3187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dumitru I., McNeil J. B. A simple in vivo footprinting method to examine DNA-protein interactions over the yeast PYK UAS element. Nucleic Acids Res. 1994 Apr 25;22(8):1450–1455. doi: 10.1093/nar/22.8.1450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Favaloro J., Treisman R., Kamen R. Transcription maps of polyoma virus-specific RNA: analysis by two-dimensional nuclease S1 gel mapping. Methods Enzymol. 1980;65(1):718–749. doi: 10.1016/s0076-6879(80)65070-8. [DOI] [PubMed] [Google Scholar]
  9. Henry Y. A., López M. C., Gibbs J. M., Chambers A., Kingsman S. M., Baker H. V., Stanway C. A. The yeast protein Gcr1p binds to the PGK UAS and contributes to the activation of transcription of the PGK gene. Mol Gen Genet. 1994 Nov 15;245(4):506–511. doi: 10.1007/BF00302263. [DOI] [PubMed] [Google Scholar]
  10. Holland M. J., Yokoi T., Holland J. P., Myambo K., Innis M. A. The GCR1 gene encodes a positive transcriptional regulator of the enolase and glyceraldehyde-3-phosphate dehydrogenase gene families in Saccharomyces cerevisiae. Mol Cell Biol. 1987 Feb;7(2):813–820. doi: 10.1128/mcb.7.2.813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Huie M. A., Scott E. W., Drazinic C. M., Lopez M. C., Hornstra I. K., Yang T. P., Baker H. V. Characterization of the DNA-binding activity of GCR1: in vivo evidence for two GCR1-binding sites in the upstream activating sequence of TPI of Saccharomyces cerevisiae. Mol Cell Biol. 1992 Jun;12(6):2690–2700. doi: 10.1128/mcb.12.6.2690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jin S. G., Prusti R. K., Roitsch T., Ankenbauer R. G., Nester E. W. Phosphorylation of the VirG protein of Agrobacterium tumefaciens by the autophosphorylated VirA protein: essential role in biological activity of VirG. J Bacteriol. 1990 Sep;172(9):4945–4950. doi: 10.1128/jb.172.9.4945-4950.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Klein C., Struhl K. Protein kinase A mediates growth-regulated expression of yeast ribosomal protein genes by modulating RAP1 transcriptional activity. Mol Cell Biol. 1994 Mar;14(3):1920–1928. doi: 10.1128/mcb.14.3.1920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Parkinson J. S., Kofoid E. C. Communication modules in bacterial signaling proteins. Annu Rev Genet. 1992;26:71–112. doi: 10.1146/annurev.ge.26.120192.000443. [DOI] [PubMed] [Google Scholar]
  16. Pavlović B., Hörz W. The chromatin structure at the promoter of a glyceraldehyde phosphate dehydrogenase gene from Saccharomyces cerevisiae reflects its functional state. Mol Cell Biol. 1988 Dec;8(12):5513–5520. doi: 10.1128/mcb.8.12.5513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sanders D. A., Gillece-Castro B. L., Stock A. M., Burlingame A. L., Koshland D. E., Jr Identification of the site of phosphorylation of the chemotaxis response regulator protein, CheY. J Biol Chem. 1989 Dec 25;264(36):21770–21778. [PubMed] [Google Scholar]
  18. Santangelo G. M., Tornow J. Efficient transcription of the glycolytic gene ADH1 and three translational component genes requires the GCR1 product, which can act through TUF/GRF/RAP binding sites. Mol Cell Biol. 1990 Feb;10(2):859–862. doi: 10.1128/mcb.10.2.859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Schleif R. DNA looping. Annu Rev Biochem. 1992;61:199–223. doi: 10.1146/annurev.bi.61.070192.001215. [DOI] [PubMed] [Google Scholar]
  20. Scott E. W., Baker H. V. Concerted action of the transcriptional activators REB1, RAP1, and GCR1 in the high-level expression of the glycolytic gene TPI. Mol Cell Biol. 1993 Jan;13(1):543–550. doi: 10.1128/mcb.13.1.543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Shore D., Nasmyth K. Purification and cloning of a DNA binding protein from yeast that binds to both silencer and activator elements. Cell. 1987 Dec 4;51(5):721–732. doi: 10.1016/0092-8674(87)90095-x. [DOI] [PubMed] [Google Scholar]
  22. Tornow J., Santangelo G. M. Efficient expression of the Saccharomyces cerevisiae glycolytic gene ADH1 is dependent upon a cis-acting regulatory element (UASRPG) found initially in genes encoding ribosomal proteins. Gene. 1990 May 31;90(1):79–85. doi: 10.1016/0378-1119(90)90441-s. [DOI] [PubMed] [Google Scholar]
  23. Tornow J., Zeng X., Gao W., Santangelo G. M. GCR1, a transcriptional activator in Saccharomyces cerevisiae, complexes with RAP1 and can function without its DNA binding domain. EMBO J. 1993 Jun;12(6):2431–2437. doi: 10.1002/j.1460-2075.1993.tb05897.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Uemura H., Jigami Y. Role of GCR2 in transcriptional activation of yeast glycolytic genes. Mol Cell Biol. 1992 Sep;12(9):3834–3842. doi: 10.1128/mcb.12.9.3834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Zhang J., Corden J. L. Identification of phosphorylation sites in the repetitive carboxyl-terminal domain of the mouse RNA polymerase II largest subunit. J Biol Chem. 1991 Feb 5;266(4):2290–2296. [PubMed] [Google Scholar]

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