BIOL 366 Lecture 15: Gene Expression in Eukaryotes I

BIOL 366 Lecture 15: Gene Expression in Eukaryotes I

• Lecture covers the yeast Gal operon and its applications, plus regulatory mechanisms unique to eukaryotes.
• Text section is 21.1.1–21.3.
• Slides will be updated before the Tuesday lecture.

Announcements

• Exam III is on November 22nd, with a review/questions on November 20th.
• Exam is cumulative, covering lectures 1–15.
• Exam format is like midterm II, including multiple-choice and written questions.
• Exam is 60 minutes long, similar to midterm II.
• Review will be next Tuesday.
• Two to three more assignments will be given on remaining lectures.
• Final exam is cumulative, with 50% of the final grade based on material after Exam II and 50% before Exam II, weighting each lecture equally.

Eukaryotic Promoters

• Eukaryotic promoters vary in complexity.
• More complex organisms have more complex promoters and control elements.
• Changes in gene expression occur during development, intercellular communication, and interaction with the environment.
• Mammalian gene promoters range from -50 to -10 kilobases (-kbp) to +10 to +50 kbp.
• Yeast gene promoters range from -250 to +1.

Eukaryotic RNA Polymerases

• All three eukaryotic RNA polymerases have little to no affinity for their promoters.
• They almost always require activator and coactivator proteins.

Regulatory Proteins

• General transcription factors are always required.
• DNA-binding transcription activators (transactivators) are important.
• DNA-binding transcription coactivators also play a role.
• Repressors are used in some cases.
• TFIID is a crucial general transcription factor.

Activation of Eukaryotic Genes

• General (basal) transcription factors are proteins required for every Pol II promoter.
• They bind the promoter region near the gene.
• Example: TATA binding Protein (TBP).
• Upstream Activating Sequences (UAS) are regulatory sites in yeast.
• Activators bind distant regulatory sites (enhancers in higher eukaryotes), recruiting Pol II complex to the promoter.
• Example: Gal4 in yeast.
• Coactivators function as bridges between activators and RNA Pol II.
• They do not directly interact with DNA.
• Example: Mediator and TFIID.
• High-Mobility Group (HMG) proteins bend DNA, leading to looping between enhancers and promoters, their binding is non-specific.

Repression of Eukaryotic Genes

• Repressors bind specific DNA sequences and inhibit transcription.
• They may disrupt contacts between Pol II and activators or coactivators.

Yeast GAL Expression System

• GAL genes allow yeast to take in and metabolize galactose.
• There are four structural Gal genes (GAL1, GAL10, GAL2, and GAL7) located in different genome loci, controlled by the same regulatory proteins.
• There are three regulatory genes:
  • GAL4: DNA-binding transcription activator (transactivator)
  • GAL80: Repressor
  • GAL3: Ligand (galactose) sensor
• GAL gene promoters consist of a TATA box and one or more upstream activator sequences (UASGAL).
• UASGAL site is recognized by the Gal4 protein, a DNA-binding transactivator.

Roles of Gal3p, Gal4p, and Gal80p

• Gal4p binds UASGAL; Gal80p prevents Gal4p's activation of Pol II in the absence of galactose.
• Gal3p, activated by galactose, binds Gal80p, changing its conformation, and inactivating it.
• This releases Gal4p, allowing it to bind Pol II, activating transcription.

Yeast Two-hybrid Assay

• A method to detect protein-protein interactions using Gal4 protein and yeast GAL genes.
• A reporter gene is placed under GAL promoter control.
• DNA-binding domain of Gal4 is fused to protein X; RNA polymerase-activating domain fused to protein Y.
• If proteins X and Y interact, DNA-binding and activation domains of Gal4 come together, activating the GAL promoter.

Other Transcription Factors

• Some transcription factors (TFs) form functional heterodimers.
• Most eukaryotic TFs bind DNA as homodimers.
• However, structurally related eukaryotic transcription factors can form heterodimers.
• Hypothetical family of four different but related proteins could form up to 10 different dimeric species.
• Each dimeric species may have unique activity.
• Example: mammalian AP-1 TFs (e.g., Fos, Jun) control cell proliferation and differentiation.
• AP-1 family members have basic leucine zipper types for protein dimerization and DNA binding.

Mobility Shift Assay

• Used to determine if a transcription factor (TF-x) binds to a promoter region of a gene (gene-Y).
• Purified TF-x is mixed with labeled genomic DNA fragment from gene-Y, run on a polyacrylamide gel, and visualized.
• Changes in DNA mobility indicate TF-x binding.

Two Regulatory Mechanisms Unique to Eukaryotes

• Genetic Imprinting: Some eukaryotes turn expression of specific alleles from one parent on and off. It is an epigenetic process based on methylation of cytosine residues and is independent from Mendelian inheritance.
• Dosage Compensation: Maintaining equal gene expression levels between sexes with different sized chromosomes (XX females and XY males). Three mechanisms exist that deal with this regulation.