Download 9.4 DNA-Binding Proteins

Chapter 9 DNA-Protein Interactions in
Bacteria
Student learning outcomes:
• Describe examples of structure /function
relationships in phage repressors
• Appreciate that altered specificity repressors and
operator mutants clarify mechanisms of amino
acid: base pair recognition
Impt. Figs. 1*, 2, 3, 4, 6, 7, 8,
14, 16, 17
Q: 1, 2, 3, 4, 5, 7, 10, 11
Cro binding DNA
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The l Family of Repressors: l, 434, P22
• Repressors have recognition
helices that lie in major groove
of appropriate operator
• Helix-turn-helix motif (HTH)
• Specificity of bp binding
depends on amino acids in
recognition helices
• Phages are not immune to
super-infection by each other
Fig. 2
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Binding Specificity of lambda-like
Repressor: Operator DNA
• Recognition helices fit sideways in major groove of
operator DNA
• Certain amino acids on DNA side of recognition helix
2 make specific contact with bases in operator
• Contacts determine specificity of protein-DNA binding
• ** Changing amino acids can change specificity of
repressor to different DNA sequence
Fig. 1
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Probing Binding Specificity by SiteDirected Mutagenesis - Mark Ptashne
Key amino acids in recognition helices of
P22, 434 repressors proposed
Amino acids differ between repressorss
Change 5 aa of 434 to P22; see altered
specificity repressor binds P22 DNA
Fig. 4
DNase footprint
Fig. 3
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l Repressor
 l repressor has extra motif, Nterminal arm that aids binding by
embracing DNA
• Cro and l repressors share affinity
for same operators, but microspecificities for OR1(l) or OR3 (cro)
• Specificities determined by
interactions between different
amino acids in recognition helices
and different base pairs in operators
Fig. 4
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l repressor dimer on OR2
High-Resolution Analysis of l
Repressor-Operator co-crystal
– Recognition helices (3 red) of
each monomer nestle into DNA
major grooves (2 half-sites)
– Helices hold two monomers
together in repressor dimer
– DNA is similar to B-form DNA
– DNA bends at ends of fragment
as curves around repressor
dimer
Fig. 6; operator sequence;
Fig. 7 model
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Amino acids of l repressor hydrogen bond
with Bases in major groove
Fig. 8
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Amino Acid: DNA Backbone Interactions
• Hydrogen bond at Gln33
maximizes electrostatic
attraction between
positively charged amino
end of a-helix and
negatively charged DNA
• Attraction works to
stabilize bond
Fig. 9
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High-Resolution Analysis of 434
Repressor-Operator Interactions
• Genetic and biochemical data
predicted R-O contacts
• X-ray crystallography of 434
repressor-fragment/ operatorfragment shows H bonding at
Gln residues in recognition helix
to 3 bp in DNA
• Potential van der Waals contact
between Gln29 and 5Me of T3
Fig. 10
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Phage 434: Effects on DNA Conformation
• R-O complex DNA deviates from normal shape
• DNA bends to accommodate base /aa contacts
• Central part of helix is wound extra tightly
• Outer parts are wound more loosely than normal
• DNA sequence of operator facilitates bending
Fig. 11 Normal DNA;
DNA bent by 434
repressor binding
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9.2 trp Repressor and role of Tryptophan
• trp repressor uses helix-turn-helix (HTH) DNA
binding motif to contact operator
• Aporepressor is not active in binding DNA
• Tryptophan forces recognition helices of trp repressor
dimer into proper position to bind trp operator
Fig. 12
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9.3 General Considerations on Protein-DNA
Interactions; multimeric proteins
• Specificity of binding between protein and specific
stretch of DNA relates to:
– Specific interactions between bases and amino acids
– Ability of DNA to assume shape that directly relates to
DNA’s base sequence
• Target sites for DNA-binding proteins usually
symmetric or repeated
• Most DNA-binding proteins are dimers: greatly
enhances binding between DNA and protein as
protein subunits bind cooperatively
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Hydrogen Bonding
Capabilities of
Different Base Pairs
• Protein ‘reads the DNA’
• Different base pairs
present four different
hydrogen-bonding
profiles to amino acids
approaching either major
or minor groove
Fig. 14
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9.4 DNA-Binding Proteins:
Action at a Distance
• DNA-binding proteins can influence interactions at
remote sites in DNA – often looping intervening DNA
• Common in eukaryotes
• Occurs in several prokaryote systems:
lac operon multiple operators
ara operon looping
gal operon looping
l repressor
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E. coli gal Operon
• gal operon has 2 operators,
97 bp apart
– One adjacent to gal promoter External operator, OE
– Other located within first
structural gene, galE Oi
Fig. 15
• 2 separated operators - both
bind repressors that interact,
loop out intervening DNA
• Recall Chapt. 7 lacI, araC
repressors
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DNA Looping affects
DNase Susceptibility
Fig. 16
Operators separated by
– Integral number of doublehelical turns loop out DNA
to allow cooperative
binding
– Nonintegral number of
turns requires proteins to
bind opposite faces of DNA,
no cooperative binding
Fig. 17 l repressor binds
cooperatively to operators
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Enhancers
Enhancers are nonpromoter DNA elements that bind
protein factors and stimulate transcription
– Can act at a distance
– Originally found in eukaryotes (lots chapt. 12)
– Recently found in prokaryotes. E. coli glnA gene:
• NtrC protein binds enhancer,
• Binds RNAP 70 bp away
• NtrC hydrolyzes ATP,
lets RPo form.
• Insert 350 bp, see loop
Fig. 20
NtrC: RNAP
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Review questions
• 1. Draw rough diagram of helix-turn-helix domain
interacting with DNA double helix
• 2. Describe experiment that shows which amino acids
bind which base pairs in l-like phage repressors.
• 10. Explain fact that protein oligomers (dimers,
tetramers) bind better to DNA than monomeric
proteins
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