Gene Regulation in Eukaryotes

Gene Regulation in Eukaryotes

• Eukaryotic cells regulate gene expression in response to environmental cues, similar to prokaryotic cells.
• Only a subset of an organism's genes is expressed at any given time, with different cell types expressing different sets of genes (e.g., skin vs. liver cells), along with housekeeping genes common to all cells.

Differences in Gene Regulation Between Eukaryotes and Prokaryotes

• Eukaryotic genes are not organized into operons like in prokaryotes.
• Eukaryotic regulatory genes are typically not linked to the genes they regulate.
• Eukaryotic regulatory proteins often need to be transported to the nucleus.
• Eukaryotic DNA is organized into nucleosomes and chromatin.

Yeast Nomenclature

• Yeast genes are named using three letters followed by a number; wild-type alleles are in all caps and italics (e.g., G A L 4 ).

• Mutant alleles are in lowercase and italics (e.g., g a l 4 ), sometimes with a specific allele number (e.g., g a l 4

  1 ).
• Phenotypes are indicated by letters, with the first letter capitalized (e.g., Gal+ for wild-type, Gal- for g a l 4 and g a l 1 mutants).
• Protein products are written as Gal4 (or Gal4p), with mutant proteins written as Gal4- or Gal4p-.

Galactose Metabolism in Yeast

• Yeast can use galactose as its sole carbon source.
• Galactose is converted into compounds that enter the glycolysis pathway.
• Mutants like g a l 1 , g a l 7 , and g a l 10 are defective in enzymes required for galactose metabolism, making them auxotrophic for galactose.
• Galactose induces Gal1p expression, where g a l 1 codes for galactokinase.
• Regulatory mechanisms of inducible genes are studied by isolating mutants that either constitutively express genes or cannot induce them.

Genetic Analysis of Gal Mutants

• Mutants can be defective in the regulatory process.
• Measuring galactokinase enzyme activity directly dissects the system.
• Common Gal mutants include g a l 4 , g a l 80 , and g a l 81 , each with distinct phenotypes related to galactose metabolism.
• g a l 4 mutants are uninducible, while g a l 80 and g a l 81 mutants are constitutive.
• The g a l 4 mutant phenotype is recessive.
• The g a l 4 mutation lies in a new gene unlinked to G A L 1 , as shown by complementation and tetrad analysis.
• Gal4p is a positive regulator of G A L 1 .
• g a l 80 mutants constitutively express galactokinase.
• g a l 80 is recessive and unlinked to other g a l genes; G A L 80 negatively regulates Gal1p expression.
• G A L 4 positively regulates, and G A L 80 negatively regulates Gal1p expression.
• Gal4p positively regulates Gal1p expression, and Gal80p negatively regulates Gal4p expression, where galactose inhibits Gal80p function.
• Gal80p negatively regulates Gal1p expression, and Gal4p negatively regulates Gal80p, where galactose activates Gal4p.

Epistasis Analysis in Yeast

• Epistasis tests are used to determine relationships between g a l 4 and g a l 80 .
• The phenotype of the g a l 4 ; g a l 80 double mutant indicates the correct model.
• If Model 1 is correct, the double mutant is uninducible; if Model 2 is correct, the double mutant is constitutive.
• A g a l 4 ; G A L 80 strain is mated with a G A L 4 ; g a l 80 strain to obtain double mutants via tetrad analysis.
• The ratio of PD:NPD:TT = 1:1:4, as g a l 4 and g a l 80 are unlinked.
• PD tetrads are easily identified, and TT tetrads have either uninducible:constitutive:wildtype = 2:1:1 or 1:2:1.
• NPD tetrads contain two wildtype spores, and two g a l 4 ; g a l 80 double mutant spores.
• The g a l 4 ; g a l 80 double mutant is uninducible, g a l 4 is epistatic to g a l 80 , suggesting g a l 4 positively regulates g a l 1 , while g a l 80 negatively regulates g a l 4 .

Finding Alleles of Existing Mutants with Different Phenotypes

• g a l 81 mutants, like g a l 80 , are constitutive for Gal1 expression, but g a l 81 is dominant.
• It is tested whether g a l 81 mutants are constitutive in a g a l 4 background to determine the epistatic relationship between g a l 81 and g a l 4 .
• Crossing g a l 81 ⋅ G A L 4 with g a l 4 ⋅ G A L 81 results in all parental ditype tetrads, indicating tight linkage between g a l 81 and g a l 4 .
• g a l 81 maps to the coding region of the g a l 4 gene, making it an allele of g a l 4 , and is renamed g a l 4 81.
• g a l 4 81 behaves as a super-activator impervious to g a l 80 , activating Gal1p expression independently of galactose and Gal80p.
• The g a l 4 81 mutation is a missense mutation altering the Gal4p protein's amino acid sequence, preventing interaction with Gal80p.

A General Model for Regulation of Eukaryotic Gene Expression

• Upstream of the G A L 1 gene, the promoter and upstream activator sequences (UASs) are required for transcriptional activation.
• TATA-binding protein (TBP) binds to the TATA box upstream of the G A L 1 transcription start site, forming a scaffold for the RNA polymerase complex.
• The promoter contains the TATA box, spanning about 40-50 bp of DNA.
• RNA polymerase binding to TBP requires activation by a transcriptional activator (Gal4p).
• Gal4p binds to the UAS, located several hundred bp upstream from the promoter and called an enhancer.
• In the absence of galactose, Gal80p physically prevents Gal4p from activating RNAP.
• In the presence of galactose, Gal80 protein changes conformation and binds to a different region of Gal4p, allowing Gal4p to activate RNAP.
• The mutation in the g a l 4 81 allele interferes with Gal80p binding, allowing mutant Gal481p protein to recruit and activate RNAP even without galactose.

Closing Thoughts

• Galactose does not directly bind to Gal80p.
• Gal3 protein directly binds galactose, allowing the Galactose/Gal3p moiety to move into the nucleus and bind Gal80p.
• This binding of Gal3p to Gal80p facilitates moving Gal80p to a different site on Gal4p, allowing Gal4p to activate transcription.