The Gal4 transcription factor is a positive regulator of gene expression of galactose-induced genes. This protein represents a large fungal family of transcription factors, Gal4 family, which includes over 50 members in the yeast ''

Saccharomyces cerevisiae ''Saccharomyces cerevisiae'' () (brewer's yeast or baker's yeast) is a species of yeast (single-celled fungus microorganisms). The species has been instrumental in winemaking, baking, and brewing since ancient times. It is believed to have b ...

'' e.g. Oaf1, Pip2, Pdr1, Pdr3, Leu3. Gal4 recognizes genes with UAS, an

upstream activating sequence An upstream activating sequence or upstream activation sequence (UAS) is a cis-acting regulatory sequence. It is distinct from the promoter and increases the expression of a neighbouring gene. Due to its essential role in activating transcription ...

, and activates them. In yeast cells, the principal targets are GAL1 (

galactokinase Galactokinase is an enzyme (phosphotransferase) that facilitates the phosphorylation of α-D-galactose to galactose 1-phosphate at the expense of one molecule of ATP. Galactokinase catalyzes the second step of the Leloir pathway, a metabolic ...

), GAL10 (

UDP-glucose 4-epimerase The enzyme UDP-glucose 4-epimerase (), also known as UDP-galactose 4-epimerase or GALE, is a homodimeric epimerase found in bacterial, fungal, plant, and mammalian cells. This enzyme performs the final step in the Leloir pathway of galactose ...

), and GAL7 (

galactose-1-phosphate uridylyltransferase Galactose-1-phosphate uridylyltransferase (or GALT, G1PUT) is an enzyme () responsible for converting ingested galactose to glucose. Galactose-1-phosphate uridylyltransferase (GALT) catalyzes the second step of the Leloir pathway of galactose ...

), three enzymes required for galactose metabolism. This binding has also proven useful in constructing the

GAL4/UAS system The GAL4-UAS system is a biochemical method used to study gene expression and function in organisms such as the fruit fly. It is based on the finding by Hitoshi Kakidani and Mark Ptashne, and Nicholas Webster and Pierre Chambon in 1988 that Gal ...

, a technique for controlling expression in insects. In yeast, Gal4 is by default repressed by Gal80, and activated in the presence of

galactose Galactose (, '' galacto-'' + ''-ose'', "milk sugar"), sometimes abbreviated Gal, is a monosaccharide sugar that is about as sweet as glucose, and about 65% as sweet as sucrose. It is an aldohexose and a C-4 epimer of glucose. A galactose molecu ...

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.

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, and specifically recognizes UAS in GAL1 promoter.

Gal4 transactivation

Localised to the C-terminus, belongs to the nine amino acids

transactivation domain The transactivation domain or trans-activating domain (TAD) is a transcription factor scaffold domain which contains binding sites for other proteins such as transcription coregulators. These binding sites are frequently referred to as activation ...

family, 9aaTAD, together with Oaf1, Pip2, Pdr1, Pdr3, but also

p53 p53, also known as Tumor protein P53, cellular tumor antigen p53 (UniProt name), or transformation-related protein 53 (TRP53) is a regulatory protein that is often mutated in human cancers. The p53 proteins (originally thought to be, and often s ...

, E2A, MLL.

Regulation

Galactose Galactose (, '' galacto-'' + ''-ose'', "milk sugar"), sometimes abbreviated Gal, is a monosaccharide sugar that is about as sweet as glucose, and about 65% as sweet as sucrose. It is an aldohexose and a C-4 epimer of glucose. A galactose molecu ...

induces Gal4 mediated transcription albeit Glucose causes severe repression. As a part of the Gal4 regulation, inhibitory protein Gal80 recognises and binds to the Gal4 region (853-874 aa). The inhibitory protein Gal80 is sequestered by regulatory protein Gal3 in Galactose dependent manner. This allows for Gal4 to work when there is galactose.

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. In the Gal4 reverted mutant Gal4C-62 mutant, 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.

Function

Target

Transcription

The Gal4 activation function is mediated by

MED15 Mediator of RNA polymerase II transcription subunit 15, also known as Gal11, Spt13 in yeast and PCQAP, ARC105, or TIG-1 in humans is a protein encoded by the ''MED15'' gene. Function MED15 is a general transcriptional cofactor of the mediator ...

(Gal11). The Gal4 protein interacts also with other mediators of transcription as are Tra1, TAF9, and SAGA/MED15 complex.

Proteosome

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

Application

The broad use of the Gal4 is in yeast

two-hybrid screening Two-hybrid screening (originally known as yeast two-hybrid system or Y2H) is a molecular biology technique used to discover protein–protein interactions (PPIs) and protein–DNA interactions by testing for physical interactions (such as bindi ...

to screen or to assay protein-protein interactions in eukaryotic cells from yeast to human. In the

, 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).