Outline

1
Outline

• Regulatory Transcription Factors
• Types
• Domains
• Functions of these domains
• -Mechanism of Regulation by these TFs

2
Structure of Eukaryotic Transcription Factors

• Many have modular structure
• DNA-binding domain
• Transcription activating domain
• Proteins can have gt 1 of each, and they can be
  in different positions in protein.
• Many also have a dimerization domain

3
Recent data suggests SP1 actually has 4
activating domains.
4
Activation Domains

• Acidic (e.g., GAL4, 49 aa domain 11 acidic aa)
• Glutamine-rich (e.g., 2 in Sp1, 25 gln)
• Proline-rich (e.g., CTF, 84 aa domain 19 are
  proline)

5
DNA-binding domains

• Zinc containing motifs
• Zinc fingers (Sp1 and TFIIIA)
• Zinc modules (GR and other nuclear receptors)
• Modules with 2 Zinc ions and 6 cysteines (GAL4)
• Homeodomains ( 60-aa domains originally found in
  homeotic mutants)
• bZIP and 4. bHLH motifs (a highly basic
  DNA-binding domain and a dimerization domain
  (leucine zipper or helix-loop-helix)

6
Amino acid side chains in proteins can form
H-bonds to DNA bases. Critical for
sequence-specific binding to DNA.
7
3 views of C2H2 Zinc fingers
Often found as repeats in a protein. Bind in the
major groove of DNA.
8
GAL4-DNA Complex

• DNA-binding domain
• 2 Zn2 bound by 6 cysteines
• A Short a helix that docks into major groove

Dimerization domain - Coiled coil (a helices)
Fig. 12.4
9
- Homeotic mutants have wrong organs
(organ-identity mutants) - Occur in animals and
plants - Important regulatory genes
Heres looking at ..uhh..you.
antennapedia
Wild-type
10
Figure 12.9

• Homeotic genes are transcription factors!
• Have a conserved DNA-binding domain
  (Homeodomain) that resembles a helix-turn-helix
  (HTH) domain.
• Bind DNA as a monomer

11
bZIP/bHLH proteins

• Have DNA binding and dimerization domains
• DNA binding region is very basic (R and K
  residues)
• Dimerization involves Leucine Zipper or
• helix-loop-helix domain
• Can form heterodimers!!

12
Leucine Zipper

• helices form a coiled coil with interdigitating
  leucines.

13
Figure 12.11a
Crystal Structure of GCN4 (bZIP) and DNA Bound
Together
Figure 12.11a
14
Crystal Structure of GCN4 (bZIP) and DNA Bound
Together
Figure 12.11b
15
Figure 12.12a
Crystal Structure of MyoD (bHLH) and DNA Bound
Together
16
Figure 12.12b
Crystal Structure of MyoD (bHLH) and DNA Bound
Together
17
(No Transcript)
18
Mechanisms of Regulation Of Gene Expression by
Regulated TFs
19
Function of Activation Domains

• Function in recruitment of components of the
  pre-initiation complex in eukaryotes (the RNAP
  holoenzyme is recruited in prokaryotes)
• Act independent of DNA-binding domains
• Can make chimeric factors that function --- i.e.,
  combine the DNA-binding domain from one factor
  and activation domain from another and get the
  expected activity

20
Figure 12.13
Activity from a chimeric transcription factor
21
YEAST TWO-HYBRID SCREENING STRATEGY
Gal4 AD
Y
X
Gal4 DBD
HIS3/lacZ
GAL1 UAS
22
Figure 12.14
Two Models for Recruitment of Yeast Preinitiation
Complex Components
23
Figure 12.15
Acidic Activation Domains Bind TFIID
24
What basal factors are specifically recruited by
transcriptional activators such as Gal4?
25
Affinity chromatography assay for effect of
factors on transcription.
DNA construct has a GAL4 binding site upstream of
a minimal Class II promoter. It is immobilized
on an agarose bead. Proteins are added and then
the column can be washed, other proteins added,
etc.. In step (d), transcription was allowed to
proceed on the bead, and the RNA product was
analyzed by primer extension (step e).
Fig. 12.16
26
The Affinity chromatography assay shows that Gal4
promotes tight binding of TFIIB.
Factors in first incubation
TFIIB does not bind very tightly without Gal4.
From Fig. 12.18
27
GAL4 (which binds to an upstream element)

• Promotes binding of TFIIB (which promotes
  recruitment of the other factors and RNAP II).
• Probably binds directly to TFIIB (i.e., it
  doesnt work by stimulating TFIID to bind TFIIB
  tighter)
• Further work has shown that GAL4 also promotes
  assembly of the downstream factors TFIIFRNAP
  II, or TFIIE.

28
Activation from a Distance Enhancers

• There are at least 3 possible models
• Factor binding to the enhancer induces
• supercoiling
• sliding
• looping

29
Models for enhancer function
RNAP II
Basal factors
promoter
Enhancer with bound protein
Modified Fig. 12.25
30
E- enhancer Psi40- rRNA promoter
Transcription of DNAs 1-5 was tested in Xenopus
oocytes. Results good transcription from 2, 3,
and 4 (also 2 gt3 or 4) but not 5. Conclusion
Enhancer has to be on same DNA molecule, but may
be far apart. Rules out the sliding and
supercoiling models, supports looping model.
From Fig. 12.27b
31
Looping out by a prokaryotic enhancer binding
protein visualized by electron microscopy.
NtrC protein that binds enhancer and RNAP s 54
polymerase RNAP with the 54 kDa sigma factor
32
Combinatorial Transcriptionexpression/regulation
depends on the combination of elements in the
promoter
Human Metallothionine promoter
GC box MRE- metal response element BLE- enhancer
that responds to activator AP1 GRE-
Glucocorticoid response element
Fig. 12.28
33
Fig. 12.32
A model of the enhanceosome assembled at the
human IFN? gene
34
How is the Regulation Achieved
35
Figure 12.6
36
Figure 12.38
37
Figure 12.39
38
Figure 12.42
Figure 12.42
39
Posttranscriptional Regulationof Transcription
Factors
Phosphorylation Ubiquitination Acetylation Sumoyla
tion
40
How can you identify the tissue/developmental
stage-specific expression of a certain gene?
Use a reporter gene
41
(No Transcript)
42
(No Transcript)
43
(No Transcript)