Simple regulatory systems:. The GAL4 Regulon. + Glucose (= no Galactose). no Glucose + Galactose. Gal3p. Gal3p. Gal4 binds to UAS G sites and regulates genes involved in Galactose metabolism. The dissociation model. The non-dissociation model. TBP. TBP.
Simple regulatory systems:
The GAL4 Regulon
(= no Galactose)
Gal4 binds to UASG sites and
regulates genes involved in Galactose metabolism
The dissociation model
The non-dissociation model
Active Gal4 protein recruits TfIId
Chromatin Immunoprecipitation (“chromatin-IP” or “chIP”)
Proteins cross-linked with
to sheared DNA
Antibody binds to
Naked DNA, ~ 200 bp average
Amplified DNA detectable by
TBP binding to the GAL1 TATA box requires an activator
(an example for chIP: chromatin Immunoprecipitation)
Gal4 protein is modular
Domain swapping proves modularity
Most transcriptional activators have independent domains
An transcription factor may use a
co-activator or co-repressor
Co-regulator = common term for either co-activator or co-repressor
The Mediator Complex
TFIId requires activators for binding to DNA
Problem: Something is missing…
Problem I: Highly purified Gal4 + RNA Polymerase II + all general transcription
factors (= GTF’s) failed to initiate transcription in vitro.
Problem II:Overexpressing Gal4 (an activator) in yeast reduced the activating
strength of other activators, a phenomenon called “squelching”.
This suggested that Gal4 was recruiting a limiting factor in yeast
cells and thus interfered with the other activator.
Experiments in crude yeast extracts showed that adding GTF’s
or RNA polymerase II did not relieve squelching.
This squelching was independent of the Gal4 DNA-binding domain,
but required the activating region of Gal4.
The missing component turns out to be a
giant multi-protein complex: the Mediator
Mediator: Major Findings
Mediator interacts with the CTD tail of RNA Pol II and stimulates TFIIH to
initiate CTD phosphorylation.
Once all yeast Mediator proteins were identified, it turned out that 13 of the
corresponding genes were already known because they were identified in
genetic screens that affect transcriptional activation and repression.
The most dramatic mutation corresponds to a yeast gene encoding Med17
(aka SRB4). This mutation is temperature-sensitive and yeast cells raised at the
permissive temperature survive. Once shifted to the restrictive temperature,
transcription of ALL Pol II-dependent genes in yeast is massively affected.
The activator-bypass experiment
DNA-binding domains: Let’s look at DNA first
The minor groove harbors little chemical Information
The Helix-Turn-Helix motif was reinvented multiple times
Anbanandam A et al. PNAS 2006;103:17225-17230
Basic Region Leucine zipper
Dimer of two large a-helices
that form a coiled coil
FOS and JUN
Helix-loop-Helix domain (bHLH)
The C2H2 zinc finger domain
Nuclear Receptors bind DNA via a pair of C4 zinc fingers
Four cysteines are complexed with a Zn++ ion
Structural properties of zinc fingers binding to DNA
C4 type (nuclear receptors)
Dimerization domains often occur in combinations