Simple regulatory systems:
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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.

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The GAL4 Regulon

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The gal4 regulon

Simple regulatory systems:

The GAL4 Regulon


The gal4 regulon

+ Glucose

(= no Galactose)

no Glucose

+ Galactose

Gal3p

Gal3p


The gal4 regulon

Gal4 binds to UASG sites and

regulates genes involved in Galactose metabolism


The gal4 regulon

The dissociation model


The gal4 regulon

The non-dissociation model


The gal4 regulon

TBP

TBP

Active Gal4 protein recruits TfIId

TfIId

no Galactose

GAL1

UASG

TATA

+ Galactose

(no Glucose)

GAL1

UASG

TATA


The gal4 regulon

Chromatin Immunoprecipitation (“chromatin-IP” or “chIP”)

Proteins cross-linked with

Formaldehyde (FA)

to sheared DNA

Antibody binds to

specific protein

Enrichment of

protein-DNA complexes

Naked DNA, ~ 200 bp average

Amplified DNA detectable by

Standard gel-electrophoresis


The gal4 regulon

DNA ladder

TBP


The gal4 regulon

TBP binding to the GAL1 TATA box requires an activator

(an example for chIP: chromatin Immunoprecipitation)


The gal4 regulon

Activation Domains


The gal4 regulon

Gal4 protein is modular


The gal4 regulon

Domain swapping proves modularity


The gal4 regulon

Most transcriptional activators have independent domains


The gal4 regulon

An transcription factor may use a

co-activator or co-repressor

Co-regulator = common term for either co-activator or co-repressor


The gal4 regulon

Activation Domains

  • In contrast to DNA-binding domains, activation domains (ADs) often do not have a conserved three-dimensional structure and are not well defined.

  • Rather, activation domains are defined by amino acid

  • content and often contain small repeats. For instance, Gal4 AD is rich in acidic and hydrophobic amino acids.

  • The potency of an activation domain depends on its overall charge, resulting in “sticky” surfaces. This variation from domain to domain and allows fine tuning of interactions between ADs and Mediator and/or GTFs.


The gal4 regulon

The Mediator Complex


The gal4 regulon

TFIId requires activators for binding to DNA

Step 1

Step 3

Step 2


The gal4 regulon

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 gal4 regulon

The missing component turns out to be a

giant multi-protein complex: the Mediator


The gal4 regulon

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 gal4 regulon

The activator-bypass experiment


The gal4 regulon

  • DNA-binding Domains

  • Structural considerations of the DNA double helix

  • Families of DNA-binding proteins


The gal4 regulon

DNA-binding domains: Let’s look at DNA first


The gal4 regulon

The minor groove harbors little chemical Information

G:C

C:G

A:T

T:A


The gal4 regulon

Helix-Turn-Helix


The gal4 regulon

The Helix-Turn-Helix motif was reinvented multiple times

Homeodomain

(yeast MatA1)

TEA domain

(human TEF-1)

Anbanandam A et al. PNAS 2006;103:17225-17230


The gal4 regulon

Basic Region Leucine zipper

(bZIP)

Dimer of two large a-helices

that form a coiled coil

Examples:

FOS and JUN


The gal4 regulon

Basic Region

Helix-loop-Helix domain (bHLH)

Example: MyoD


The gal4 regulon

The C2H2 zinc finger domain

Example: TFIIIA


The gal4 regulon

Nuclear Receptors bind DNA via a pair of C4 zinc fingers

Four cysteines are complexed with a Zn++ ion


The gal4 regulon

Structural properties of zinc fingers binding to DNA

C2H2 type

C4 type (nuclear receptors)


The gal4 regulon

Dimerization domains often occur in combinations


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