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					     Footprinting DNA-Protein
            Interactions
•  Powerful and fairly rapid methods for
   mapping where and how proteins bind
   tightly to DNA
• 2 examples:
  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. 5.36a
Sample of a DNase I       Fig. 5.36b
footprinting gel (for a
DNA-binding protein).




            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 DNA fragments on
   gel.



   Fig. 5.37a
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. 5.37b
  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, i.e., lack of activation instead of true
       repression
   – Cells respond to high glucose with low 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           (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
                    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 -   carboxy-terminal domain                       Fig. 7.19
NTD -   amino-terminal domain
     CAP-cAMP bends the activator DNA




Similar to 7.17b
Bending may promote interaction of RNAP with
       a distal upstream element (U).
                 Or …

• Bending the DNA simply increases the
  DNA surface in contact with CAP-
  cAMP, giving greater binding.
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) & Co-
    repressor (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-->
Transcription stops in the leader-attenuator “L” region
when the [tryptophan] is elevated.
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?
                     REGULON

•     Collection of genes not in the same operon that are
      co-regulated

•     Examples:
1.    SOS response regulon: DNA repair genes induced
      by DNA damaging agents

2.    Maltose (mal) regulon: genes needed to metabolize
      maltose (glucose-glucose)
     – Involves multiple promoters regulated by:
       • MalT (a regulatory protein that requires
               inducer, maltotriose)
       • MalT and CAP
       • CAP alone
          The mal Regulon
• Encodes genes needed for maltose utilization
• Involves multiple promoters, some regulated
  by:
   – MalT (a regulatory protein that also
      requires ATP and the mal regulon
      inducer, maltotriose)
   – MalT and CAP
   – CAP alone
 Regulation Summary (so far)
• Operon
  – Co-transcription (all)
  – Repression (Lac, Trp)
  – Attenuation (Trp)
  – Activation (Lac)
• Regulon
  – Multiple promoters and operons
  – Some share an activator (MalT, CAP)

				
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