11.4 - Combinatorial control in eukaryotes (copy)

what are GAL genes in Yeast?

Genes that are needed for eukaryotic cell to be able to use galactose if glucose is not available

Upstream activating sequence (UAS)

• The equivalent in yeast of the enhancer in higher eukaryotes;

• it is bound by transcriptional activator proteins.

GAL genes

• location

• regulation

• Many genes scattered over many chromosomes – no operons like in bacteria – but still regulated coordinately by common set of proteins

• All GAL genes have similar promoters and are regulated by common set of proteins

5 levels of regulation

• Chromatin opening (SWI/SNF, acetylation)

• Non-coding RNA transcripts (turn off expression @ promoter)

• UAS has both enhancer and Mig1 repressor binding sites (silencer)

• GAL-specific induction system

• Catabolite repression

how are Yeast GAL Genes activated and repressed?

• GAL1/10 genes are positively regulated by the activator Gal4 protein

• GAL1/10 genes are negatively regulated by a noncoding RNA synthesized from a cryptic promoter that controls chromatin structure.

in which direction is GAL1 transcribed? in which direction is GAL10 transcribed?

• what protein binds to UAS?

• GAL1 is transcribed to the right

  • promoter region is 118 bp long and contains 4 upstream activator sites (UAS)

• GAL10 is transcribed in the opposite direction from the same control region

• UAS bind DNA-binding trans-activator protein made by GAL4 gene – Gal4p (Gal4 protein)

trans-activator Gal4p

binds to UAS in front of many GAL genes

Turn on GAL genes - describe the proteins involved

• Need Gal4p – transcription activator – needed to bind to RNA polymerase to start transcription of genes so cell can use galactose

• involves additional proteins besides GAL4p

  • The inhibitor – Gal80p

  • Ligand sensor – Gal3p

    • senses presence of galactose

  • Mig1 –presence in nucleus dependent on phosphorylation – dependent on absence of glucose

  • Tup1 – binds to Mig1 – blocks transcription

Yeast GAL Genes: A Model for Activation and Repression

• what is Gal4p’s role

• how do Gal4p, Gal80p and Gal3p interact?

• what is catabolite repression mediated by?

• Gal4 is negatively regulated by Gal80.

• Gal80 is negatively regulated by Gal3, the ultimate positive regulator, which is activated by the inducer, galactose (the ligand).

• Activated Gal4 recruits the machinery necessary to alter the chromatin and recruit RNA polymerase.

• Catabolite repression is mediated by a glucose-dependent protein kinase, Snf1

GAL-specific induction system - GALACTOSE Absent

Gal80p made

• Binds to Gal4p and blocks Gal4p from binding to RNA polymerase to help start transcription therefore no transcription

GALACTOSE Present

Galactose binds to Gal3p which binds to ATP

• Activated Gal3p binds to Gal80p, causes a conformational change in Gal80p so it no longer blocks Gal4p from binding to RNA polymerase therefore transcription occurs

Catabolite Repression

• when both glucose and galactose are absent do we get transcription?

• when glucose is absent but galactose is present, do we get transcription?

• no

• yes

Catabolite Repression - Both sugars are present

Glucose keeps GAL genes turned off even if galactose is present, HOW?

• Normally Mig1 is in cytoplasm in phosphorylated state and can not enter nucleus BUT if glucose is present phosphorylation is inhibited and Mig1 enters nucleus and binds to DNA site.

• Tup1 then can bind to Mig1 and blocks transcription, may also cause histone deacetylation which closes chromatin structure

How important are multiple UAS sites?

• what happens when one binding site is deleted?

• what happens when both binding sites are deleted?

• what happens when a binding site is mutated?

• what happens when a mutation occurs before the binding sites?

• still have transcription, just less

• no transcription

• still have transcription, just less

• normal transcription (no effect)