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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

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chapter 9 dna protein interactions in bacteria
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

the l family of repressors l 434 p22
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

binding specificity of lambda like repressor operator dna
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

probing binding specificity by site directed mutagenesis mark ptashne
Probing Binding Specificity by Site-Directed 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

l repressor
l Repressor
  • l repressor has extra motif, N-terminal arm that aids binding by embracing DNA
  • Cro and l repressors share affinity for same operators, but micro-specificities 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

l repressor dimer on OR2

high resolution analysis of l repressor operator co crystal
High-Resolution Analysis of lRepressor-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

amino acid dna backbone interactions
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

high resolution analysis of 434 repressor operator interactions
High-Resolution Analysis of 434 Repressor-Operator Interactions
  • Genetic and biochemical data predicted R-O contacts
  • X-ray crystallography of 434 repressor-fragment/ operator-fragment 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

phage 434 effects on dna conformation
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

9 2 trp repressor and role of tryptophan
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

9 3 general considerations on protein dna interactions multimeric proteins
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
hydrogen bonding capabilities of different base pairs
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

9 4 dna binding proteins action at a distance
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

e coli gal operon
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
  • 2 separated operators - both bind repressors that interact, loop out intervening DNA
  • Recall Chapt. 7 lacI, araC repressors

Fig. 15

dna looping affects dnase susceptibility
DNA Looping affects DNase Susceptibility

Fig. 16

Operators separated by

  • Integral number of double-helical 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

enhancers
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

review questions
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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