Footprinting DNA-Protein
Interactions
•
Powerful and fairly rapid methods for
mapping where and how proteins bind
tightly to DNA
• 2 ways:
1. DNAse I footprinting
2. DMS footprinting
DNAse I Footprinting
1. Prepare end-labeled DNA.
2. Bind protein.
3. Mild digestion with DNAse I
(randomly cleaves DS DNA
on each strand)
4. Separate DNA fragments on
denaturing acrylamide gels.
Fig. 5.37a
Fig. 5.37b
Sample of a DNase I
footprinting gel.
Footprint
Samples in lanes 2-4
had increasing amounts
of the DNA-binding
protein (lambda protein
cII); lane 1 had none.
Dimethylsulfate (DMS) Footprinting
1. End-label DNA fragment.
2. Bind protein.
3. Treat with DMS, methylates
purines.
4. Partially cleave DNA at the
methylated bases.
5. Separate fragments on gel.
Fig. 5.38a
Example of DMS footprinting.
Lanes 1 and 4 had no protein
Lanes 2 and 3 had 2 different
amounts of protein.
Protein binding protects most purines
from modification by DMS, but it can
stimulate modification of those in
regions where the helix is distorted or
partially melted (indicated by *) .
Fig. 5.38b
Positive Control of Lac Operon
• Catabolite Repression hypothesis
– predicted that glucose would inhibit synthesis of
other sugar metabolizing pathway enzymes (e.g.,
lactose pathway)
• Partially right, its lack of activation instead of true
repression
– Cells respond to high glucose with lowered levels
of cAMP and vice-versa
– cAMP activates Lac operon via CAP
cyclic 5’-3’ phosphodiester in cAMP
glucose
cAMP
- Stimulates Lac operon (lacZ production)
as the co-activator for the CAP protein
CRP bends -->
CAP (catabolite activator protein), a.k.a.
crp (cAMP receptor protein) gene
•
•
CAP only active bound to cAMP
CAP-cAMP stimulates transcription by promoting
formation of closed complex:
RNAP + Pro ↔ RPc → RPo
Kb
k2
(RPc = Closed complex)
(RPo = Open complex)
Kb – equilibrium binding constant for formation of RPc
k2 – rate constant for formation of RPo
•
CAP-cAMP increases Kb
Lac Control Region
• CAP binds just upstream of promoter
• L1 deletion mutant has constitutively low expression
Fig. 7.16
CAP-cAMP dimer interacts with the CTD of the
a subunits of the RNAP Core
CAP-cAMP is a dimer that binds to a short sequence
(~20 bp) with dyad symmetry (activator site)
αCTD binds DNA too
CTD NTD -
carboxy-terminal domain
amino-terminal domain
Fig. 7.19
CAP-cAMP-aCTD and CAP-cAMP-DNA complexes:
CAP-cAMP bends the activator DNA
Fig 7.17
Why does the Lac Operon need an activator?
Not a very good core promoter:
-35
-10
TTTACAC ---------------- TATGTT
(Lac)
-35
-10
TTGACAT --------------- TATAAT (consensus)
CAP stimulates more than 100 promoters!
Tryptophan operon: Regulation
by attenuation
•
•
•
•
Genes for tryptophan synthesis
Repressed by end-product of pathway,
Tryptophan
Repression requires Operator sequence,
Aporepressor (trpR gene product) & Corepressor (Tryptophan)
- Operator is within the promoter
Also controlled by attenuation in the
“Leader” region of the transcript
Low [tryptophan], aporepressor doesn’t bind Operator,
transcription on!
High [tryptophan], repressor (aporep. + tryp.) binds
operator, represses transcription!
Attenuation-->
The trp operon is also controlled by attenuation.
P
P/O L
E
D
C
B
140 bp
Moderate trp
AUG
7000 bp
Low trp
AUG
Transcription stops in the leader-attenuator “L” region
when the [tryptophan] is elevated.
A
The trp Leader peptide (14 aa) has two key tryptophan codons.
The ribosome stalls at the trp codons when [tryptophan] is
too low. The stalled ribosome prevents a downstream
transcription terminator (IR + U-rich sequence) from
forming.
Fig. 7.31
Fig. 7.32
Biological advantage:
• Repression alone decreases expression 70-fold
• Repression plus attenuation decreases
expression 700-fold
How is translation of the downstream genes
achieved with the leader peptide there to stop
the ribosomes?