Chapter 9 dna protein interactions in bacteria
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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

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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 acids of l repressor hydrogen bond with bases in major groove

Amino acids of l repressor hydrogen bond with Bases in major groove

Fig. 8


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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