Chapter 9 DNAProtein Interactions in Prokaryotes

1
Chapter 9 DNA-Protein Interactions in
Prokaryotes

• 
  
• 
  
  2006-10-18 Yao Qi

2

• From chapter 7 and 8 we leant that several
  proteins can bind tightly to specific sites on
  DNA. (lac repressor , trp repressor, ?repressor,
  Cro, CAP)

3

• All mentioned five proteins have a similar
  structure motif helix-turn-helix motif. This
  motif allows the recognition helix to fit snugly
  into the major groove of of the target DNA site.

Figure 9.1
4

• In this chapter well discuss several
  well-studied examples of specific DNA-protein
  interactions in prokaryotic cells to see what
  makes them so specific.

5

• 9.1 The?Family of Repressors
• 9.2 The trp Repressor
• 9.3 General Considerations on
• Protein- DNA Interactions
• 9.4 DNA-Binding ProteinsAction at a
• Distance

6
9.1 The ? Family of Repressors

• Box 9.1 X-Ray Crystallography
• High-Resolution Analysis of ? Repressor-Operator
  Interactions
• High-Resolution Analysis of Phage 434
  Repressor-Operator Interactions

7
Introductions to the ? family repressors

• The repressors of ? and similar phages have
  recognition helices that lie in the major groove
  of the appropriate operators as shown in Figure
  9.1 and 9.2 .

Figure 9.2
Back
8

• ?- like phages 434 P22
• 434 and P22 have very similar molecular
  genetics, but they have different immunity
  regions they make different repressors that
  recognize different operators.

9

• Immunity
• An E.coli cell lysogenized by ? phage is
  immune to superinfection by ? because the excess
  ? repressors in the lysogen immediately bind to
  the superinfection ? DNA and prevents its
  expression.

10

• Using x-ray diffraction analysis of
  operator-repressor complex, they have identified
  the face of the recognition helix of the 434
  phage repressor that contacts the bases in the
  major groove of its operator. By analogy, one
  could make a similar prediction for the P22
  repressor.

11
This picture illustrates the amino acids in each
repressor that are most likely to be involved in
operator binding.
12

• Changing these amino acids can change the
  specificity of the repressor?

13

• In vivo
• Construct recombinant 434 repressor with 5 amino
  acids in the recognition helix changed to match
  those in the phage P22.
• Transform E.coli cells with this recombinant 434
  repressor. (Actually, 434 and P22 do not infect
  E.coli cells, so they used recombinant ? phage
  with the 434 immunity regions)

14

• Result
• The cells producing the altered 434 repressor
  were immune to infection by the ? phage with the
  P22 immunity region, but not to infection by the
  ? phage with the 434 immunity region.

15

• In vitro
• DNase footprinting
• 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.

16

• DNase footprinting with end-labeled P22
  phage OR and either P22 repressor or the
  recombinant 434 repressor .
• The two sets of lanes contained increasing
  concentrations of the respective repressors (0 M
  in lanes 1 and 8, and ranging from 7.610-10 M to
  1.110-8 M in lanes 2-7 and from 5.210-9 M to
  5.710-7 M in lanes 8-14.

DNase footprinting with the recombinent 434
repressor
17

• The binding specificity really had been
  altered by these five amino acid changes.
• conclusion
• specific interactions between bases and amino
  acids defines the specificity between a protein
  and a specific stretch of DNA

18
High-Resolution Analysis of ? Repressor-Operator
Interactions

• X-Ray Crystallography
• This the central instrument to get the
  structure information of protein, DNA,
  protein-DNA complex , through which we can find
  out how protein interact with DNA.

19

• Using partial molecules to get better cystals
  which can achieve a resolution of 25 Å.
• The repreesor fragment encompassed residues
  1-92,which include all of the DNA-binding
  domainof the protein.
• The operator fragment was 20 bp long and
  contained one complete site to which the
  repressor dimer attached.( two half-sites , each
  bound to a repressor monomer)

20
General structure features

• Its a more detailed representation of figure9.2
  model . It revealed several general aspects of
  the protein-DNA interaction.
• Recognition helices of each repressor monomer
  nestled into the major groove in the two-half
  sites.
• Helices 5 5 approach each other to hold the
  two monomers together in the repressor dimer.
• There is a bit of binding of the DNA as it curves
  around the repressor dimer.

21
How the DNA-protein recognition works

• Amino acid interactions with bases
• Figure 9.8 shows the details of the
  interactions between amino acids in a repressor
  monomer and bases in one operator half-site.
• Amino acid/DNA backbone interactions
• Figure 9.9 shows amino acid/DNA backbone
  interactions

22
Figure 9.8
Back
23
Figure 9.9

• This diagram is perpendicular to that in Figure
  9.8 .
• a-helices 1-4 of the ? repressor are shown, along
  with the phosphates (PA-PE) that are involved in
  hydrogen bonds with the protein

24

• Hydrogen bond network
• amino acid base
• amino acid DNA bone phosphate
• side chains on amino acid DNA bone phosphate
• amino acid amino acid

25
Conformation of biochemical and genetic data

• 1. Ethylation of certain operator phosphates
  interfered with repressor binding.
• 2. Methylation protection experiment certain
  guanines in the major groove would be in close
  contact with repressor.
• 3. DNA sequence data A-T at position 2 , G-C at
  position 4 were conserved in all 12 half-site of
  the operators OR and OL.
• 4. Genetic data mutations in certain amino acids
  destabilized repressor-operator ineractions,
  while other changes in aa enhanced binding to the
  operator.
• All these are in almost complete agreement
  with the structure derived from the crystal .

26
High-Resolution Analysis of Phage 434
Repressor-Operator Interactions

• The x-ray crystallography analysis of the partial
  repressor complex shows that
• Close approaches between certain amino acids and
  certain phosphate in the operators DNA backbone.
• Hydrogen bonding between aa base pairs.
• Potential van der Waals contact.

27

• The DNA deviates significantly from its normal
  regular shape. It bends somewhat to accommodate
  the necessary base/amino acid contacts. Moreover
  , the central part of the helix, between the two
  half-sites, is wound tightly, and the outer parts
  are wound more loosely than normal.
• The base sequence of the operator facilitate
  these departures from normal DNA shape.

28
(No Transcript)
29
9.2 The trp Repressor

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

30

• The trp repressor binds to the trp operator in a
  less-directed way than the ? family of repressors
  bind to their operators. The recognition helix of
  the trp repressor points directly into the major
  groove of the operator, which allows only one
  direct contact between an amino acid and a base.

31
What contributes to the specificity between a
protein and s specific stretch of DNA?

• 1. specific interactions between bases and amino
  acids
• 2. the ability of the DNA to assume a certain
  shape (depends on the DNA sequence)

32
9.3 General Considerations on Protein-DNA
Interactions

• Hydrogen Bonding Capalilities of the Four
  Different Base Pairs
• The Role of DNA Shape in Specific Binding to
  Proteins
• The Importance of Multimeric DNA-Binding Proteins

33
Hydrogen Bonding Capabilities of the Four
Different Base Pairs
34
The Importance of Multimeric DNA-Binding Proteins

• Enhance the binding between DNA and protein
  because the protein subunits bind cooperatively.
  This boost in concentration is important for
  DNA-binding proteins are generally present in the
  cell in very small quantities.
• Entropy use the least energy to from the
  DNA-protein complex.

35
9.4 DNA-Binding Proteins Action at a
Distance

• The gal Operon
• Duplicated ? Operators
• The lac Operon
• Enhancers

36
The gal Operon
37
Duplicated ? Operators

• Artificial system
• Separate the adjacent operators to varying
  extents (integral number of helical turns and
  nonintegral number of helical turns).
• To find out whether the repressor dimers
  still bind cooperatively when separated . Check
  by DNase footprinting and electron microscopy.

38
Affect of DNA looping on DNase susceptibility
39

Cooperatively binding
NonCooperatively binding
40
(No Transcript)
41
(No Transcript)
42

• When ? operators are separated by an integral
  number of helical turns, the DNA in between can
  loop out to allow cooperative binding.
• When the operators are separated by a
  nonintegral number of helical turns, the proteins
  have to bind to opposite faces of the DNA double
  helix, so no cooperative binding can take place.

43
The lac Operon

• The lac operon has three operators one major
  operator near the transcription start site, and
  two auxiliary operators, one upstream and one
  downsream.
• They act cooperatively in repression.

44

• Construct DNA fragment containing 2 copies of the
  classical lac operator placed 200bp apart.
• Use three different assays for looping
• electron microscopy
• DNase footprinting
• gel electrophoresis

45
DNA looping between two lac operators
46
Enhancers

• Enhancers are nonpromoter DNA elements that bind
  protein factors and stimulate transcription. By
  definition, they can act at a distance.

47

• E.coli glnA gene
• NtrC protein enhancer interact by looping
  out the DNA in between.
• electron microscopy
• phage T4
• Late enhancer phage DNA-replication .

48

• Moved the glnA promoter and enhancer by inserting
  a 350bp DNA segment between them. Add Ntrc (bind
  to the enhancer )and RNA polymerase (bind to the
  promoter).

49

• Thank you!