Gal4 transcription factor

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{{missing information|full narrative of how it works in yeast; other targets like GAL7|date=April 2019}}}}

{{infobox nonhuman protein|Organism=Saccharomyces cerevisiae|Symbol=GAL4|Name=Regulatory protein GAL4|UniProt=P04386|EntrezGene=855828}}

The Gal4 transcription factor is a positive regulator of gene expression of galactose-induced genes.{{cite journal | vauthors = Klar AJ, Halvorson HO | title = Studies on the positive regulatory gene, GAL4, in regulation of galactose catabolic enzymes in Saccharomyces cerevisiae | journal = Molecular & General Genetics | volume = 135 | issue = 3 | pages = 203–12 | year = 1974 | pmid = 4376212 | doi = 10.1007/BF00268616| s2cid = 26014344 }} 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.{{cite journal | vauthors = Schjerling P, Holmberg S | title = Comparative amino acid sequence analysis of the C6 zinc cluster family of transcriptional regulators | journal = Nucleic Acids Research | volume = 24 | issue = 23 | pages = 4599–607 | date = December 1996 | pmid = 8967907 | pmc = 146297 | doi = 10.1093/nar/24.23.4599}}

Gal4 recognizes genes with UAS{{sub|G}}, 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.{{cite journal | vauthors = Duffy JB | title = GAL4 system in Drosophila: a fly geneticist's Swiss army knife | journal = Genesis | volume = 34 | issue = 1–2 | pages = 1–15 | date = 2002 | pmid = 12324939 | doi = 10.1002/gene.10150 | s2cid = 5073328 | doi-access = free }} In yeast, Gal4 is by default repressed by Gal80, and activated in the presence of galactose as Gal3 binds away Gal80.

Domains

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

Image:Gal4 9aaTAD.jpg

=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,{{cite journal | vauthors = Marmorstein R, Carey M, Ptashne M, Harrison SC | title = DNA recognition by GAL4: structure of a protein-DNA complex | journal = Nature | volume = 356 | issue = 6368 | pages = 408–14 | date = April 1992 | pmid = 1557122 | doi = 10.1038/356408a0 | bibcode = 1992Natur.356..408M | s2cid = 4344434 }}{{cite journal | vauthors = Pan T, Coleman JE | title = The DNA binding domain of GAL4 forms a binuclear metal ion complex | journal = Biochemistry | volume = 29 | issue = 12 | pages = 2023–9 | date = March 1990 | pmid = 2186803 | doi = 10.1021/bi00464a019 }} and specifically recognizes UAS{{sub|G}} in GAL1 promoter. {{cite journal | vauthors = Keegan L, Gill G, Ptashne M | title = Separation of DNA binding from the transcription-activating function of a eukaryotic regulatory protein | journal = Science | volume = 231 | issue = 4739 | pages = 699–704 | date = February 1986 | pmid = 3080805 | doi = 10.1126/science.3080805 | bibcode = 1986Sci...231..699K }}{{cite journal | vauthors = Giniger E, Varnum SM, Ptashne M | title = Specific DNA binding of GAL4, a positive regulatory protein of yeast | journal = Cell | volume = 40 | issue = 4 | pages = 767–74 | date = April 1985 | pmid = 3886158 | doi = 10.1016/0092-8674(85)90336-8 | s2cid = 31663066 }}

=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.{{cite journal | vauthors = Ding WV, Johnston SA | title = The DNA binding and activation domains of Gal4p are sufficient for conveying its regulatory signals | journal = Molecular and Cellular Biology | volume = 17 | issue = 5 | pages = 2538–49 | date = May 1997 | pmid = 9111323 | pmc = 232103 | doi = 10.1128/MCB.17.5.2538}}{{cite journal | vauthors = Melcher K, Johnston SA | title = GAL4 interacts with TATA-binding protein and coactivators | journal = Molecular and Cellular Biology | volume = 15 | issue = 5 | pages = 2839–48 | date = May 1995 | pmid = 7739564 | pmc = 230515 | doi = 10.1128/MCB.15.5.2839}}

Regulation

Galactose induces Gal4 mediated transcription albeit Glucose causes severe repression.{{cite journal | vauthors = Klar AJ, Halvorson HO | title = Studies on the positive regulatory gene, GAL4, in regulation of galactose catabolic enzymes in Saccharomyces cerevisiae | journal = Molecular & General Genetics | volume = 135 | issue = 3 | pages = 203–12 | year = 1974 | pmid = 4376212 | doi = 10.1007/BF00268616| s2cid = 26014344 }}{{cite journal | vauthors = Griggs DW, Johnston M | title = Regulated expression of the GAL4 activator gene in yeast provides a sensitive genetic switch for glucose repression | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 88 | issue = 19 | pages = 8597–601 | date = October 1991 | pmid = 1924319 | pmc = 52556 | doi = 10.1073/pnas.88.19.8597| bibcode = 1991PNAS...88.8597G | doi-access = free }}

As a part of the Gal4 regulation, inhibitory protein Gal80 recognises and binds to the Gal4 region (853-874 aa).{{cite journal | vauthors = Kumar PR, Yu Y, Sternglanz R, Johnston SA, Joshua-Tor L | title = NADP regulates the yeast GAL induction system | journal = Science | volume = 319 | issue = 5866 | pages = 1090–2 | date = February 2008 | pmid = 18292341 | pmc = 2726985 | doi = 10.1126/science.1151903 | bibcode = 2008Sci...319.1090K }}{{cite journal | vauthors = Thoden JB, Ryan LA, Reece RJ, Holden HM | title = The interaction between an acidic transcriptional activator and its inhibitor. The molecular basis of Gal4p recognition by Gal80p | journal = The Journal of Biological Chemistry | volume = 283 | issue = 44 | pages = 30266–72 | date = October 2008 | pmid = 18701455 | pmc = 2573081 | doi = 10.1074/jbc.M805200200 | doi-access = free }}{{cite journal | vauthors = Johnston SA, Salmeron JM, Dincher SS | title = Interaction of positive and negative regulatory proteins in the galactose regulon of yeast | journal = Cell | volume = 50 | issue = 1 | pages = 143–6 | date = July 1987 | pmid = 3297350 | doi = 10.1016/0092-8674(87)90671-4| s2cid = 46090047 }}

The inhibitory protein Gal80 is sequestered by regulatory protein Gal3 in Galactose dependent manner. This allows for Gal4 to work when there is galactose.{{cite journal | vauthors = Egriboz O, Jiang F, Hopper JE | title = Rapid GAL gene switch of Saccharomyces cerevisiae depends on nuclear Gal3, not nucleocytoplasmic trafficking of Gal3 and Gal80 | journal = Genetics | volume = 189 | issue = 3 | pages = 825–36 | date = November 2011 | pmid = 21890741 | pmc = 3213366 | doi = 10.1534/genetics.111.131839 }}{{cite journal | vauthors = Jiang F, Frey BR, Evans ML, Friel JC, Hopper JE | title = Gene activation by dissociation of an inhibitor from a transcriptional activation domain | journal = Molecular and Cellular Biology | volume = 29 | issue = 20 | pages = 5604–10 | date = October 2009 | pmid = 19651897 | pmc = 2756894 | doi = 10.1128/MCB.00632-09 }}{{cite journal | vauthors = Peng G, Hopper JE | title = Gene activation by interaction of an inhibitor with a cytoplasmic signaling protein | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 99 | issue = 13 | pages = 8548–53 | date = June 2002 | pmid = 12084916 | pmc = 124307 | doi = 10.1073/pnas.142100099 | bibcode = 2002PNAS...99.8548P | doi-access = free }}{{cite journal | vauthors = Tsuyumu S, Adams BG | title = Dilution kinetic studies of yeast populations: in vivo aggregation of galactose utilizing enzymes and positive regulator molecules | journal = Genetics | volume = 77 | issue = 3 | pages = 491–505 | date = July 1974 | doi = 10.1093/genetics/77.3.491 | pmid = 4369925 | pmc = 1213142 }}

=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.{{cite journal | vauthors = Douglas HC, Condie F | title = The genetic control of galactose utilization in Saccharomyces | journal = Journal of Bacteriology | volume = 68 | issue = 6 | pages = 662–70 | date = December 1954 | pmid = 13221541 | pmc = 386212 | doi = 10.1128/jb.68.6.662-670.1954}}{{cite journal | vauthors = Douglas HC, Hawthorne DC | title = Enzymatic Expression and Genetic Linkage of Genes Controlling Galactose Utilization in Saccharomyces | journal = Genetics | volume = 49 | pages = 837–44 | date = May 1964 | issue = 5 | doi = 10.1093/genetics/49.5.837 | pmid = 14158615 | pmc = 1210618 }}{{cite journal | vauthors = Matsumoto K, Adachi Y, Toh-e A, Oshima Y | title = 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 = Journal of Bacteriology | volume = 141 | issue = 2 | pages = 508–27 | date = February 1980 | pmid = 6988385 | pmc = 293654 | doi = 10.1128/JB.141.2.508-527.1980}}

In the Gal4 reverted mutant Gal4C-62 mutant,{{cite journal | vauthors = Johnston SA, Salmeron JM, Dincher SS | title = Interaction of positive and negative regulatory proteins in the galactose regulon of yeast | journal = Cell | volume = 50 | issue = 1 | pages = 143–6 | date = July 1987 | pmid = 3297350 | doi = 10.1016/0092-8674(87)90671-4| s2cid = 46090047 }} 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.{{cite journal | vauthors = Ma J, Ptashne M | title = Deletion analysis of GAL4 defines two transcriptional activating segments | journal = Cell | volume = 48 | issue = 5 | pages = 847–53 | date = March 1987 | pmid = 3028647 | doi = 10.1016/0092-8674(87)90081-X| s2cid = 4979320 }}{{cite journal | vauthors = Warfield L, Tuttle LM, Pacheco D, Klevit RE, Hahn S | title = A sequence-specific transcription activator motif and powerful synthetic variants that bind Mediator using a fuzzy protein interface | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 111 | issue = 34 | pages = E3506-13 | date = August 2014 | pmid = 25122681 | pmc = 4151740 | doi = 10.1073/pnas.1412088111 | bibcode = 2014PNAS..111E3506W | doi-access = free }}

Function

=Target=

=Transcription=

The Gal4 activation function is mediated by MED15 (Gal11).{{cite journal | vauthors = Fassler JS, Winston F | title = The Saccharomyces cerevisiae SPT13/GAL11 gene has both positive and negative regulatory roles in transcription | journal = Molecular and Cellular Biology | volume = 9 | issue = 12 | pages = 5602–9 | date = December 1989 | pmid = 2685570 | pmc = 363730 | doi = 10.1128/MCB.9.12.5602}}{{cite journal | vauthors = Han Y, Kodadek T | title = Peptides selected to bind the Gal80 repressor are potent transcriptional activation domains in yeast | journal = The Journal of Biological Chemistry | volume = 275 | issue = 20 | pages = 14979–84 | date = May 2000 | pmid = 10809742 | doi = 10.1074/jbc.275.20.14979| doi-access = free }}{{cite journal | vauthors = Hashimoto H, Kikuchi Y, Nogi Y, Fukasawa T | title = Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae. Isolation and characterization of the regulatory gene GAL4 | journal = Molecular & General Genetics | volume = 191 | issue = 1 | pages = 31–8 | year = 1983 | pmid = 6350827 | doi = 10.1007/BF00330886| s2cid = 115216273 }}{{cite journal | vauthors = Long RM, Mylin LM, Hopper JE | title = GAL11 (SPT13), a transcriptional regulator of diverse yeast genes, affects the phosphorylation state of GAL4, a highly specific transcriptional activator | journal = Molecular and Cellular Biology | volume = 11 | issue = 4 | pages = 2311–4 | date = April 1991 | pmid = 2005915 | pmc = 359938 | doi = 10.1128/MCB.11.4.2311}}{{cite journal | vauthors = Nogi Y, Fukasawa T | title = A novel mutation that affects utilization of galactose in Saccharomyces cerevisiae | journal = Current Genetics | volume = 2 | issue = 2 | pages = 115–20 | date = October 1980 | pmid = 24189802 | doi = 10.1007/BF00420623 | s2cid = 12635991 }}{{cite journal | vauthors = Sakurai H, Hiraoka Y, Fukasawa T | title = Yeast GAL11 protein is a distinctive type transcription factor that enhances basal transcription in vitro | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 90 | issue = 18 | pages = 8382–6 | date = September 1993 | pmid = 8378310 | pmc = 47360 | doi = 10.1073/pnas.90.18.8382| bibcode = 1993PNAS...90.8382S | doi-access = free }}{{cite journal | vauthors = Suzuki Y, Nogi Y, Abe A, Fukasawa T | title = GAL11 protein, an auxiliary transcription activator for genes encoding galactose-metabolizing enzymes in Saccharomyces cerevisiae | journal = Molecular and Cellular Biology | volume = 12 | issue = 10 | pages = 4806 | date = October 1992 | pmid = 1406662 | pmc = 360407 | doi = 10.1128/MCB.12.10.4806}}

The Gal4 protein interacts also with other mediators of transcription as are Tra1,{{cite journal | vauthors = Lin L, Chamberlain L, Zhu LJ, Green MR | title = Analysis of Gal4-directed transcription activation using Tra1 mutants selectively defective for interaction with Gal4 | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 109 | issue = 6 | pages = 1997–2002 | date = February 2012 | pmid = 22308403 | pmc = 3277556 | doi = 10.1073/pnas.1116340109 | url = http://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=1180&context=pgfe_pp | bibcode = 2012PNAS..109.1997L | doi-access = free }}{{cite journal | vauthors = Majmudar CY, Labut AE, Mapp AK | title = Tra1 as a screening target for transcriptional activation domain discovery | journal = Bioorganic & Medicinal Chemistry Letters | volume = 19 | issue = 14 | pages = 3733–5 | date = July 2009 | pmid = 19497740 | pmc = 4322765 | doi = 10.1016/j.bmcl.2009.05.045 }}{{cite journal | vauthors = Reeves WM, Hahn S | title = Targets of the Gal4 transcription activator in functional transcription complexes | journal = Molecular and Cellular Biology | volume = 25 | issue = 20 | pages = 9092–102 | date = October 2005 | pmid = 16199885 | pmc = 1265783 | doi = 10.1128/MCB.25.20.9092-9102.2005 }} TAF9,{{cite journal | vauthors = Klein J, Nolden M, Sanders SL, Kirchner J, Weil PA, Melcher K | title = Use of a genetically introduced cross-linker to identify interaction sites of acidic activators within native transcription factor IID and SAGA | journal = The Journal of Biological Chemistry | volume = 278 | issue = 9 | pages = 6779–86 | date = February 2003 | pmid = 12501245 | doi = 10.1074/jbc.M212514200 | doi-access = free }} and SAGA/MED15 complex.{{cite journal | vauthors = Larschan E, Winston F | title = The Saccharomyces cerevisiae Srb8-Srb11 complex functions with the SAGA complex during Gal4-activated transcription | journal = Molecular and Cellular Biology | volume = 25 | issue = 1 | pages = 114–23 | date = January 2005 | pmid = 15601835 | pmc = 538787 | doi = 10.1128/MCB.25.1.114-123.2005 }} (http://mcb.asm.org/content/25/1/114/F8.large.jpg){{cite journal | vauthors = Larsson M, Uvell H, Sandström J, Rydén P, Selth LA, Björklund S | title = Functional studies of the yeast med5, med15 and med16 mediator tail subunits | journal = PLOS ONE | volume = 8 | issue = 8 | pages = e73137 | year = 2013 | pmid = 23991176 | pmc = 3750046 | doi = 10.1371/journal.pone.0073137 | bibcode = 2013PLoSO...873137L | doi-access = free }}

=Proteosome=

A subunit of the 26 S proteasome Sug2 regulatory protein has a molecular and functional interaction with Gal4 function.{{cite journal | vauthors = Chang C, Gonzalez F, Rothermel B, Sun L, Johnston SA, Kodadek T | title = The Gal4 activation domain binds Sug2 protein, a proteasome component, in vivo and in vitro | journal = The Journal of Biological Chemistry | volume = 276 | issue = 33 | pages = 30956–63 | date = August 2001 | pmid = 11418596 | doi = 10.1074/jbc.M102254200 | doi-access = free }}{{cite journal | vauthors = Russell SJ, Johnston SA | title = Evidence that proteolysis of Gal4 cannot explain the transcriptional effects of proteasome ATPase mutations | journal = The Journal of Biological Chemistry | volume = 276 | issue = 13 | pages = 9825–31 | date = March 2001 | pmid = 11152478 | doi = 10.1074/jbc.M010889200 | doi-access = free }} Proteolytic turnover of the Gal4 transcription factor is not required for function in vivo.{{cite journal | vauthors = Nalley K, Johnston SA, Kodadek T | title = Proteolytic turnover of the Gal4 transcription factor is not required for function in vivo | journal = Nature | volume = 442 | issue = 7106 | pages = 1054–7 | date = August 2006 | pmid = 16929306 | doi = 10.1038/nature05067 | bibcode = 2006Natur.442.1054N | s2cid = 1926315 }} The native Gal4 monoubiquitination protects from 19S-mediated destabilizing under inducing conditions.{{cite journal | vauthors = Ferdous A, Sikder D, Gillette T, Nalley K, Kodadek T, Johnston SA | title = The role of the proteasomal ATPases and activator monoubiquitylation in regulating Gal4 binding to promoters | journal = Genes & Development | volume = 21 | issue = 1 | pages = 112–23 | date = January 2007 | pmid = 17167105 | pmc = 1759896 | doi = 10.1101/gad.1493207 }}

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.

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).