Regulatory protein GAL4
Identifiers
OrganismSaccharomyces cerevisiae
SymbolGAL4
Entrez855828
UniProtP04386
Search for
StructuresSwiss-model
DomainsInterPro

The Gal4 transcription factor is a positive regulator of gene expression of galactose-induced genes.[1] This protein represents a large fungal family of transcription factors, Gal4 family, which includes over 50 members in the yeast Saccharomyces cerevisiae e.g. Oaf1, Pip2, Pdr1, Pdr3, Leu3.[2]

Gal4 recognizes genes with UASG, an upstream activating sequence, and activates them. In yeast cells, the principal targets are GAL1 (galactokinase), GAL10 (UDP-glucose 4-epimerase), and GAL7 (galactose-1-phosphate uridylyltransferase), three enzymes required for galactose metabolism. This binding has also proven useful in constructing the GAL4/UAS system, a technique for controlling expression in insects.[3] In yeast, Gal4 is by default repressed by Gal80, and activated in the presence of galactose as Gal3 binds away Gal80.[4]

Domains

Two executive domains, DNA binding and activation domains, provide key function of the Gal4 protein conforming to most of the transcription factors.

Gal4 domains and regulation

DNA binding

Gal4 N-terminus is a zinc finger and belongs to the Zn(2)-C6 fungal family. It forms a Zn – cysteines thiolate cluster,[5][6] and specifically recognizes UASG in GAL1 promoter. [7][8]

Gal4 transactivation

Localised to the C-terminus, belongs to the nine amino acids transactivation domain family, 9aaTAD, together with Oaf1, Pip2, Pdr1, Pdr3, but also p53, E2A, MLL.[9][10]

Regulation

Galactose induces Gal4 mediated transcription albeit Glucose causes severe repression.[11][12]

As a part of the Gal4 regulation, inhibitory protein Gal80 recognises and binds to the Gal4 region (853-874 aa).[13][14][15]

The inhibitory protein Gal80 is sequestered by regulatory protein Gal3 in Galactose dependent manner. This allows for Gal4 to work when there is galactose.[16][4][17][18]

Mutants

The Gal4 loss-of-function mutant gal4-64 (1-852 aa, deletion of the Gal4 C-terminal 29 aa) lost both interaction with Gal80 and activation function.[19][20][21]

In the Gal4 reverted mutant Gal4C-62 mutant,[22] a sequence (QTAY N AFMN) with the 9aaTAD pattern emerged and restored activation function of the Gal4 protein.

Inactive constructs

The activation domain Gal4 is inhibited by C-terminal domain in some Gal4 constructs.[23][24]

Function

Target

Transcription

The Gal4 activation function is mediated by MED15 (Gal11).[25][26][27][28][29][30][31]

The Gal4 protein interacts also with other mediators of transcription as are Tra1,[32][33][34] TAF9,[35] and SAGA/MED15 complex.[36][37]

Proteosome

A subunit of the 26 S proteasome Sug2 regulatory protein has a molecular and functional interaction with Gal4 function.[38][39] Proteolytic turnover of the Gal4 transcription factor is not required for function in vivo.[40] The native Gal4 monoubiquitination protects from 19S-mediated destabilizing under inducing conditions.[41]

Application

The broad use of the Gal4 is in yeast two-hybrid screening to screen or to assay protein-protein interactions in eukaryotic cells from yeast to human.

In the GAL4/UAS system, the Gal4 protein and Gal4 upstream activating region (UAS) are used to study the gene expression and function in organisms such as the fruit fly.[3]

The Gal4 and inhibitory protein Gal80 have found application in a genetics technique for creating individually labeled homozygous cells called MARCM (Mosaic analysis with a repressible cell marker).

See also

References

  1. ↑ Klar AJ, Halvorson HO (1974). "Studies on the positive regulatory gene, GAL4, in regulation of galactose catabolic enzymes in Saccharomyces cerevisiae". Molecular & General Genetics. 135 (3): 203–12. doi:10.1007/BF00268616. PMID 4376212. S2CID 26014344.
  2. ↑ Schjerling P, Holmberg S (December 1996). "Comparative amino acid sequence analysis of the C6 zinc cluster family of transcriptional regulators". Nucleic Acids Research. 24 (23): 4599–607. doi:10.1093/nar/24.23.4599. PMC 146297. PMID 8967907.
  3. 1 2 Duffy JB (2002). "GAL4 system in Drosophila: a fly geneticist's Swiss army knife". Genesis. 34 (1–2): 1–15. doi:10.1002/gene.10150. PMID 12324939. S2CID 5073328.
  4. 1 2 Jiang F, Frey BR, Evans ML, Friel JC, Hopper JE (October 2009). "Gene activation by dissociation of an inhibitor from a transcriptional activation domain". Molecular and Cellular Biology. 29 (20): 5604–10. doi:10.1128/MCB.00632-09. PMC 2756894. PMID 19651897.
  5. ↑ Marmorstein R, Carey M, Ptashne M, Harrison SC (April 1992). "DNA recognition by GAL4: structure of a protein-DNA complex". Nature. 356 (6368): 408–14. Bibcode:1992Natur.356..408M. doi:10.1038/356408a0. PMID 1557122. S2CID 4344434.
  6. ↑ Pan T, Coleman JE (March 1990). "The DNA binding domain of GAL4 forms a binuclear metal ion complex". Biochemistry. 29 (12): 2023–9. doi:10.1021/bi00464a019. PMID 2186803.
  7. ↑ Keegan L, Gill G, Ptashne M (February 1986). "Separation of DNA binding from the transcription-activating function of a eukaryotic regulatory protein". Science. 231 (4739): 699–704. Bibcode:1986Sci...231..699K. doi:10.1126/science.3080805. PMID 3080805.
  8. ↑ Giniger E, Varnum SM, Ptashne M (April 1985). "Specific DNA binding of GAL4, a positive regulatory protein of yeast". Cell. 40 (4): 767–74. doi:10.1016/0092-8674(85)90336-8. PMID 3886158. S2CID 31663066.
  9. ↑ Ding WV, Johnston SA (May 1997). "The DNA binding and activation domains of Gal4p are sufficient for conveying its regulatory signals". Molecular and Cellular Biology. 17 (5): 2538–49. doi:10.1128/MCB.17.5.2538. PMC 232103. PMID 9111323.
  10. ↑ Melcher K, Johnston SA (May 1995). "GAL4 interacts with TATA-binding protein and coactivators". Molecular and Cellular Biology. 15 (5): 2839–48. doi:10.1128/MCB.15.5.2839. PMC 230515. PMID 7739564.
  11. ↑ Klar AJ, Halvorson HO (1974). "Studies on the positive regulatory gene, GAL4, in regulation of galactose catabolic enzymes in Saccharomyces cerevisiae". Molecular & General Genetics. 135 (3): 203–12. doi:10.1007/BF00268616. PMID 4376212. S2CID 26014344.
  12. ↑ Griggs DW, Johnston M (October 1991). "Regulated expression of the GAL4 activator gene in yeast provides a sensitive genetic switch for glucose repression". Proceedings of the National Academy of Sciences of the United States of America. 88 (19): 8597–601. Bibcode:1991PNAS...88.8597G. doi:10.1073/pnas.88.19.8597. PMC 52556. PMID 1924319.
  13. ↑ Kumar PR, Yu Y, Sternglanz R, Johnston SA, Joshua-Tor L (February 2008). "NADP regulates the yeast GAL induction system". Science. 319 (5866): 1090–2. Bibcode:2008Sci...319.1090K. doi:10.1126/science.1151903. PMC 2726985. PMID 18292341.
  14. ↑ Thoden JB, Ryan LA, Reece RJ, Holden HM (October 2008). "The interaction between an acidic transcriptional activator and its inhibitor. The molecular basis of Gal4p recognition by Gal80p". The Journal of Biological Chemistry. 283 (44): 30266–72. doi:10.1074/jbc.M805200200. PMC 2573081. PMID 18701455.
  15. ↑ Johnston SA, Salmeron JM, Dincher SS (July 1987). "Interaction of positive and negative regulatory proteins in the galactose regulon of yeast". Cell. 50 (1): 143–6. doi:10.1016/0092-8674(87)90671-4. PMID 3297350. S2CID 46090047.
  16. ↑ Egriboz O, Jiang F, Hopper JE (November 2011). "Rapid GAL gene switch of Saccharomyces cerevisiae depends on nuclear Gal3, not nucleocytoplasmic trafficking of Gal3 and Gal80". Genetics. 189 (3): 825–36. doi:10.1534/genetics.111.131839. PMC 3213366. PMID 21890741.
  17. ↑ Peng G, Hopper JE (June 2002). "Gene activation by interaction of an inhibitor with a cytoplasmic signaling protein". Proceedings of the National Academy of Sciences of the United States of America. 99 (13): 8548–53. Bibcode:2002PNAS...99.8548P. doi:10.1073/pnas.142100099. PMC 124307. PMID 12084916.
  18. ↑ Tsuyumu S, Adams BG (July 1974). "Dilution kinetic studies of yeast populations: in vivo aggregation of galactose utilizing enzymes and positive regulator molecules". Genetics. 77 (3): 491–505. doi:10.1093/genetics/77.3.491. PMC 1213142. PMID 4369925.
  19. ↑ Douglas HC, Condie F (December 1954). "The genetic control of galactose utilization in Saccharomyces". Journal of Bacteriology. 68 (6): 662–70. doi:10.1128/jb.68.6.662-670.1954. PMC 386212. PMID 13221541.
  20. ↑ Douglas HC, Hawthorne DC (May 1964). "Enzymatic Expression and Genetic Linkage of Genes Controlling Galactose Utilization in Saccharomyces". Genetics. 49 (5): 837–44. doi:10.1093/genetics/49.5.837. PMC 1210618. PMID 14158615.
  21. ↑ Matsumoto K, Adachi Y, Toh-e A, Oshima Y (February 1980). "Function of positive regulatory gene gal4 in the synthesis of galactose pathway enzymes in Saccharomyces cerevisiae: evidence that the GAL81 region codes for part of the gal4 protein". Journal of Bacteriology. 141 (2): 508–27. doi:10.1128/JB.141.2.508-527.1980. PMC 293654. PMID 6988385.
  22. ↑ Johnston SA, Salmeron JM, Dincher SS (July 1987). "Interaction of positive and negative regulatory proteins in the galactose regulon of yeast". Cell. 50 (1): 143–6. doi:10.1016/0092-8674(87)90671-4. PMID 3297350. S2CID 46090047.
  23. ↑ Ma J, Ptashne M (March 1987). "Deletion analysis of GAL4 defines two transcriptional activating segments". Cell. 48 (5): 847–53. doi:10.1016/0092-8674(87)90081-X. PMID 3028647. S2CID 4979320.
  24. ↑ Warfield L, Tuttle LM, Pacheco D, Klevit RE, Hahn S (August 2014). "A sequence-specific transcription activator motif and powerful synthetic variants that bind Mediator using a fuzzy protein interface". Proceedings of the National Academy of Sciences of the United States of America. 111 (34): E3506-13. Bibcode:2014PNAS..111E3506W. doi:10.1073/pnas.1412088111. PMC 4151740. PMID 25122681.
  25. ↑ Fassler JS, Winston F (December 1989). "The Saccharomyces cerevisiae SPT13/GAL11 gene has both positive and negative regulatory roles in transcription". Molecular and Cellular Biology. 9 (12): 5602–9. doi:10.1128/MCB.9.12.5602. PMC 363730. PMID 2685570.
  26. ↑ Han Y, Kodadek T (May 2000). "Peptides selected to bind the Gal80 repressor are potent transcriptional activation domains in yeast". The Journal of Biological Chemistry. 275 (20): 14979–84. doi:10.1074/jbc.275.20.14979. PMID 10809742.
  27. ↑ Hashimoto H, Kikuchi Y, Nogi Y, Fukasawa T (1983). "Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae. Isolation and characterization of the regulatory gene GAL4". Molecular & General Genetics. 191 (1): 31–8. doi:10.1007/BF00330886. PMID 6350827. S2CID 115216273.
  28. ↑ Long RM, Mylin LM, Hopper JE (April 1991). "GAL11 (SPT13), a transcriptional regulator of diverse yeast genes, affects the phosphorylation state of GAL4, a highly specific transcriptional activator". Molecular and Cellular Biology. 11 (4): 2311–4. doi:10.1128/MCB.11.4.2311. PMC 359938. PMID 2005915.
  29. ↑ Nogi Y, Fukasawa T (October 1980). "A novel mutation that affects utilization of galactose in Saccharomyces cerevisiae". Current Genetics. 2 (2): 115–20. doi:10.1007/BF00420623. PMID 24189802. S2CID 12635991.
  30. ↑ Sakurai H, Hiraoka Y, Fukasawa T (September 1993). "Yeast GAL11 protein is a distinctive type transcription factor that enhances basal transcription in vitro". Proceedings of the National Academy of Sciences of the United States of America. 90 (18): 8382–6. Bibcode:1993PNAS...90.8382S. doi:10.1073/pnas.90.18.8382. PMC 47360. PMID 8378310.
  31. ↑ Suzuki Y, Nogi Y, Abe A, Fukasawa T (October 1992). "GAL11 protein, an auxiliary transcription activator for genes encoding galactose-metabolizing enzymes in Saccharomyces cerevisiae". Molecular and Cellular Biology. 12 (10): 4806. doi:10.1128/MCB.12.10.4806. PMC 360407. PMID 1406662.
  32. ↑ Lin L, Chamberlain L, Zhu LJ, Green MR (February 2012). "Analysis of Gal4-directed transcription activation using Tra1 mutants selectively defective for interaction with Gal4". Proceedings of the National Academy of Sciences of the United States of America. 109 (6): 1997–2002. Bibcode:2012PNAS..109.1997L. doi:10.1073/pnas.1116340109. PMC 3277556. PMID 22308403.
  33. ↑ Majmudar CY, Labut AE, Mapp AK (July 2009). "Tra1 as a screening target for transcriptional activation domain discovery". Bioorganic & Medicinal Chemistry Letters. 19 (14): 3733–5. doi:10.1016/j.bmcl.2009.05.045. PMC 4322765. PMID 19497740.
  34. ↑ Reeves WM, Hahn S (October 2005). "Targets of the Gal4 transcription activator in functional transcription complexes". Molecular and Cellular Biology. 25 (20): 9092–102. doi:10.1128/MCB.25.20.9092-9102.2005. PMC 1265783. PMID 16199885.
  35. ↑ Klein J, Nolden M, Sanders SL, Kirchner J, Weil PA, Melcher K (February 2003). "Use of a genetically introduced cross-linker to identify interaction sites of acidic activators within native transcription factor IID and SAGA". The Journal of Biological Chemistry. 278 (9): 6779–86. doi:10.1074/jbc.M212514200. PMID 12501245.
  36. ↑ Larschan E, Winston F (January 2005). "The Saccharomyces cerevisiae Srb8-Srb11 complex functions with the SAGA complex during Gal4-activated transcription". Molecular and Cellular Biology. 25 (1): 114–23. doi:10.1128/MCB.25.1.114-123.2005. PMC 538787. PMID 15601835. (http://mcb.asm.org/content/25/1/114/F8.large.jpg)
  37. ↑ Larsson M, Uvell H, Sandström J, Rydén P, Selth LA, Björklund S (2013). "Functional studies of the yeast med5, med15 and med16 mediator tail subunits". PLOS ONE. 8 (8): e73137. Bibcode:2013PLoSO...873137L. doi:10.1371/journal.pone.0073137. PMC 3750046. PMID 23991176.
  38. ↑ Chang C, Gonzalez F, Rothermel B, Sun L, Johnston SA, Kodadek T (August 2001). "The Gal4 activation domain binds Sug2 protein, a proteasome component, in vivo and in vitro". The Journal of Biological Chemistry. 276 (33): 30956–63. doi:10.1074/jbc.M102254200. PMID 11418596.
  39. ↑ Russell SJ, Johnston SA (March 2001). "Evidence that proteolysis of Gal4 cannot explain the transcriptional effects of proteasome ATPase mutations". The Journal of Biological Chemistry. 276 (13): 9825–31. doi:10.1074/jbc.M010889200. PMID 11152478.
  40. ↑ Nalley K, Johnston SA, Kodadek T (August 2006). "Proteolytic turnover of the Gal4 transcription factor is not required for function in vivo". Nature. 442 (7106): 1054–7. Bibcode:2006Natur.442.1054N. doi:10.1038/nature05067. PMID 16929306. S2CID 1926315.
  41. ↑ Ferdous A, Sikder D, Gillette T, Nalley K, Kodadek T, Johnston SA (January 2007). "The role of the proteasomal ATPases and activator monoubiquitylation in regulating Gal4 binding to promoters". Genes & Development. 21 (1): 112–23. doi:10.1101/gad.1493207. PMC 1759896. PMID 17167105.

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