Chater9 DNAprotein interactions in prokaryotes

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Chater9DNA-protein interactions in prokaryotes

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• 200722040135

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• 1?Several proteins (lac repressor, CAP, trp
  repressor, ? repressor, and Cro)can bind to one
  particular short DNA sequence among a vast excess
  of unrelated sequences.

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• 2?All five proteins have a similar structural
  motif helix-turn-helix motif- two a-helices
  connected by a short proteins turn.

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• 9.1 Some Repressors
• 1.The?Family of Repressors
• 2.The trp Repressor
• 9.2 The function of DNA in Protein- DNA
  Interactions

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9.1 The?Family of Repressors

• Characteristic
• 1.Helix-turn-helix motif
• 2.Interact with specific DNA by amino acids in
  the recognition helix
• 3.The specific binding depends on certain amino
  acids in the recognition helices

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9.1 The?Family of Repressors

• We would like to know which are important amino
  acids in these interactions.
• Experiment 1
• How do these proteins accomplish such specific
  binding?
• Experiment 2

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Experiment 1-- by Mark Ptashne and his colleagues

• Material434 P22 (The?Family of Repressors )
• Two characteristics
• Similarity helix-turn-helix motif.
• Dissimilarity they recognize different
  operators for different immunity regions.

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Experiment 1-- by Mark Ptashne and his colleagues

• Suspicions
• 1?what amino acids in the recognition helices
  contact with the bases in the DNA major groove?
  (Answer1)
• 2?If are they decide the specificity of the
  repressor? (Answer2)

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Suspicion1

• Method X-ray diffraction analysis of
  operator-repressor complex.
• They identified the face of the recognition helix
  of the 434 phage repressor that contacts the
  bases in the major groove of its operator.

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Suspicion1

• This picture illustrates the amino acids in each
  repressor that are most likely to be involved in
  operator binding.

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Suspicion1

• Conclusion
• Different repressors have different amino acids
  in the recognition helix that can interact with
  the specific DNA.

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Suspicion2

• Material
• Construct recombinant 434 repressor immunity
  regions that changed 5 amino acids in the
  recognition helix to those of phage P22.
• Method
• They expressed the altered gene in bacteria and
  tested the product for ability to bind to 434 and
  P22 operators, both in vivo and in vitro.
• Result
• In vivo
• In vitro

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Suspicion2
P22
Y

• In vivo
• The assay was to infect E.coli cells with the
  recombinant phage.

434
N
434
E.coli
P22
N
P22
Y
434
E.coli
P22
N
434
Y
E.coli
434
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Suspicion2

• Result
• The E.coli cells which are infected by
  recombinant 434 phage are immune to the P22
  superinfection, but not to 434 superinfection.

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Suspicion2

• In vitro
• Method
• DNase footprinting (picture)

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DNase footprinting with the recombinent 434
repressor

• Suspicion2

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Suspicion2

• Result
• The purified recombinant repressor could make a
  footprint in the P22 operator, just as P22
  repressor can while could no longer make a
  footprint in the 434 represor.
• Conclusion Changing these amino acids can change
  the specificity of the repressor.

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Experiment 2by Steven Jordan and Carl Pabo

• Method
• The assay used X-Ray Crystallography to perform a
  detailed analysis of the interaction between the
  repressor and operator.
• Material
• The repressor fragment (residues 1-92) include
  all of the DNA-binding domain of the protein.
• The operator fragment (20 bp) contained one
  complete repressor dimer attached site.

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Experiment 2by Steven Jordan and Carl Pabo

• How the DNA-protein recognition works?
• Steven Jordan and Carl Pabo achieve a resolution
  of 2.5 angstroms by making excellent co-crystals
  of a repressor fragment and an operator fragment.

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Experiment 2by Steven Jordan and Carl Pabo

• Hydrogen bond
• amino acid base
• amino acid DNA bone phosphate
• side chains on amino acid DNA bone phosphate
• amino acid amino acid
• Some of these H-bonds are stabilized by H-bond
  networks involving two amino acids and two or
  more sites on the DNA.

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• When they used X-ray crystallography to analyze
  the fragment of phage 434 repressor-operator
  complex, they found that it also has a potential
  van der Waals contact between an amino acid in
  the recognition helix and a base in the operator
  besides the hydrogen bonding.

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9.1 The trp Repressor

• The trp repressor consists of aporepressor and
  tryptopha.
• The Role of Tryptophan
• The tryptophan force the recognition helices of
  the repressor dimer into the proper position for
  interacting with the trp operator.
• Flash

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9.2 The function of DNA in Protein- DNA
Interactions

• The Role of DNA Shape in Specific Binding to
  Proteins

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The Role of DNA Shape in Specific Binding to
Proteins

• It not always just the amino acid-DNA base-pair
  interactions that govern the affinity between
  protein and DNA the affinity between DNA and
  protein may also depend on the ability of DNA to
  be distorted into a shape that fits the protein.
• Reason The contacts we have discussed between
  the repressor and the DNA backbone require that
  DNA double helix curve slightly, as illustrate in
  next figure.

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The Role of DNA Shape in Specific Binding to
Proteins
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• Now we know that this bend is primarily due to
  two kinks, or abrupt turns in the DNA helix, at
  which adjacent base pairs unstack and no longer
  lie parallel to each other.

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• For the kinks, the TG sequence is important, not
  because of any specific contacts it make with the
  protein, but because it allows the all important
  kink to occur.

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The Role of DNA Shape in Specific Binding to
Proteins

• The affinity between DNA and protein may depend
  on the ability of the DNA to be distorted into a
  shape that fits the protein.

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• Summarize
• The amino acids in the recognition helix
  determine the specificity of the protein that
  bind to specific DNA
• The slightly curve of the B-form DNA also
  determine the affinity between DNA-protein
  complex.
• The combine between the complex are main the
  hydrogen bonds.

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• Thank you!