Footprinting%20DNA-Protein%20Interactions

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Footprinting DNA-Protein Interactions

• Powerful and fairly rapid methods for mapping
  where and how proteins bind tightly to DNA
• 2 ways
• DNAse I footprinting
• DMS footprinting

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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
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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.
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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
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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
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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

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cyclic 5-3 phosphodiester in cAMP
glucose
cAMP
- Stimulates Lac operon (lacZ production) as the
co-activator for the CAP protein
CRP bends --gt
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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 (RPc Closed
  complex)
• Kb k2 (RPo Open
  complex)
• Kb equilibrium binding constant for formation
  of RPc
• k2 rate constant for formation of RPo
• CAP-cAMP increases Kb

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Lac Control Region

• CAP binds just upstream of promoter
• L1 deletion mutant has constitutively low
  expression

Fig. 7.16
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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)
aCTD binds DNA too
CTD - carboxy-terminal domain NTD -
amino-terminal domain
Fig. 7.19
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CAP-cAMP-?CTD and CAP-cAMP-DNA
complexesCAP-cAMP bends the activator DNA

Fig 7.17
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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!
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Tryptophan operon Regulation by attenuation

• Genes for tryptophan synthesis
• Repressed by end-product of pathway, Tryptophan
• Repression requires Operator sequence,
  Aporepressor (trpR gene product) Co- repressor
  (Tryptophan)
• - Operator is within the promoter
• Also controlled by attenuation in the Leader
  region of the transcript

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Low tryptophan, aporepressor doesnt bind
Operator, transcription on!
High tryptophan, repressor (aporep. tryp.)
binds operator, represses transcription!
Attenuation--gt
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Transcription stops in the leader-attenuator L
region when the tryptophan is elevated.
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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
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Fig. 7.32
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• 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?