Advanced Molecular Genetics-Biology 566 Eukaryotic Polymerases and the Yeast GAL gene.
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Comparison of prokaryotic and eukaryotic RNA polymerases.

Single prokaryotic RNA polymerase. Sigma factor helps in selection of promoters.

Three eukaryotic RNA polymerases.

RNA polymerase I: rRNA

RNA polymerase II: mRNA

RNA polymerase III: tRNA, 5S rRNA

Fig 10.26 Comparison of prokaryotic and eukaryotic RNA polymerase subunit homology. (Lodish et al., 2000)

What would you expect the eukaryotic organellar RNA polymerases to look like? Why?

What do promoters for RNA polymerase II look link for eukaryotic transcription initiation?

Fig 10.30 Eukaryotic promoter consensus sequence. (Lodish et al., 2000).

In yeast the TATAA is located at -85. Why so far away?

How do the subunits interact to initiate transcription at the promoter sites in eukaryotes?

Figure 10.50 Progress of transcription initiation. (Lodish et.al., 2000).

How is eukaryotic RNA polymerase II regulated?

by recruitment?

by polymerase activation?

by promoter activation?

Evidence:

- Transcription machinery is not bound to the promoter prior to activation.

- Yeast activators have separable activating and DNA-binding domains.

- An activating region must be tethered to DNA near a promoter to activate transcription.

- A given activating region will work when attached to a heterologous DNA-binding domain, even one taken from bacteria.

- Any of a wide array of genes can be brought under control of a given activator by introducing the appropriate activator binding sites nearby.

- Various "activator bypass" experiments work.

- Many different peptide sequences work as activating regions; their properties are more suggestive of adhesive surfaces than of specialized functions.

Conclusion: Recruitment is the major mechanism of eukaryotic gene transcription activation.

The Yeast GAL Genes model.

Fig. 2.1 The GAL1 gene and its upstream regulatory regions.

Fig. 2.2 Three states of the GAL1 gene.

In the absence of galactose, Gal4 binds the UASg but the activation domain is masked by Gal80.

As shown by the outline in fig 2.2c, the Gal4 protein production is also inhibited by glucose.

Phosphorylated Mig1 is held in the cytoplasm. Glucose prevents phosphorylation of Mig1 and allow it to enter the nucleus.

Generalization:
  1- Without the presence of an activation signal, transcription activation factors are held in the cytoplasm.
  2- If transcription activator is present in the nucleus it's activation domain is usually blocked or inactivated without the presence of an activation signal.

Fig. 2.3 & 2.4 Gal4 dimer bound to UAS site and it's active domains.

Fig 2.5 Testing the separate Gal4 domains for activation activity.

Fig. 2.6 Activation by modification of Gal80.

Conclusion:

- there are few, if any, sterospecific restrictions on how the activating region must attach.

- any protein-protein interaction that brings an activating region to DNA will elicit transcription.

Yeast two hybrid system.

Fig. 2.7 Yeast two-hybrid system.

What is the nature of the activator interaction?

- activating regions are acidic in addition to hydrophobic residues (similar to many other activating regions)

- mutations that eliminate positively charged residues from this domain increase the activation strength of Gal4.

- the strength of activation is proportional to the length of the activation domain.

- reiterated motifs compensate for sequence changes.

-scrambling the order of amino acids destroys the activation function.

Demonstrating that activators operate via recruitment.

Fig. 2.10 Squelching. Activator A has no DNA-binding site and when it is overproduced it sequesters all of the target. Activator B has a DNA-binding site but has no free target to bind to.

Fig 2.11. Recruitment visualized by Chromatin ImmunoPrecipitation or ChIP analysis. Shows the proteins that are bound to specific regions of the chromatin.

Fig. 2.12. Activator bypass. Activation domain of Gal4 removed but able to bind to a "P" potentiator mutation in the mediator protein Gal11. C domains function in activation even when switched.

Fig. 2.13. Activator bypass by direct tethering.