essentials of protein - nucleic acids interactions and...
TRANSCRIPT
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Essentials of protein - nucleic acids interactions and thermodynamics
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B-DNA – major & minor grooves
G & G 12.11
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Pitch 34 Å
Rise3.4 Å
Width 20 Å
Major Groove
Minor Groove
10.4 bp/turn
B-DNA: A rightHanded double helix
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Protein-DNA interaction
• Sequence independent– may interact with the negatively charged sugar-
phosphate backbone• Sequence dependent
– need to recognize the bases in the double-helical structure (don’t have access to the atoms involved in base pair H-bonds)
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DNA Polymerase III - replication of the E.coli chromosome
• At least 10 different subunits
• α is polymerase, ε is 3'-exonuclease, θ unknown function
• β dimer forms a ring around DNA – acts as sliding clamp
to hold res of PolIII in place
– sequence independent
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Histone octamer - Nucleosome
nucleosome axis
H1 linker histoneDNA core histone
histone octamer
nucleosome core particle
entry-exitangle α
H2A, H2B, H3, H4
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Nucleosome crystal structure
•Histone proteins are Lys & Arg rich – basic•Histones form octamers •Basic histones interact with the negatively charged phosphates of the DNA•The DNA is wrapped around a core formed by 4 different histone polypeptides
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Hydration of nucleosome
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Specific binding of proteins to DNA
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Which groove?
• H-bond donors and aceptors different in major groove for 2 types of base pair but very similar in the minor groove
• Minor groove too small to accommodate large protein probe
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Which groove?
• H-bond donors and aceptors different in major groove for 2 types of base pair but very similar in the minor groove
• Minor groove too small to accommodate large protein probe
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Which groove?
• H-bond donors and aceptors different in major groove for 2 types of base pair but very similar in the minor groove
• Minor groove too small to accommodate large protein probe
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Hydrogen bonding between aspargaine and adenine
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Structural motifs in DNA-binding regulatory proteins
• Crucial feature must be atomic contacts between protein residues and bases and sugar-phosphate backbone of DNA
• Most contacts are in the major groove of DNA • 80% of regulatory proteins can be assigned to one of three
classes: - helix-turn-helix (HTH)- zinc finger (Zn-finger)- leucine zipper (bZIP)- helix-loop-helix (HLH)
• In addition to DNA-binding domains, these proteins often possess other domains that interact with other proteins
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α Helices and DNA - a perfect fit
• DNA-binding proteins often have an a-helical segments that fit directly into the major groove of B-form DNA
• Diameter of helix is 1.2 nm (12 Angstroms)
– Major groove of DNA is about 1.2 nm wide and 0.6 to 0.8 nm deep
• Proteins can recognize specific sites (sequences) in DNA
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The helix-turn-helix motif
• Generally bind as dimers to dyad-symmetric sites on DNA
• All contain two alpha helices separated by a loop with a beta turn
• The C-terminal helix fits in major groove of DNA
• N-terminal helix stabilised by hydrophobic interactions with C-terminal helix
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The helix-turn-helix motif: homeodomain transcription factor
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Binding of a protein dimer to a symmetric DNA binding site
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The helix-turn-helix motif (tryptophan repressor, lambda Cro, lambda repressor, Lac repressor etc)
• Generally bind as dimers to dyad-symmetric sites on DNA
• All contain two alpha helices separated by a loop
• The C-terminal helix fits in major groove of DNA
• N-terminal helix stabilised by hydrophobic interactions with C-terminal helix
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Cys2His2 zinc finger protein family
largest knownDNA-bindingfamily inmulticellularorganisms
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Cys2His2 zinc finger: DNA binding
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Estrogen receptor binds DNA via zinc fingers
• Homodimer• 2 Zn fingers
interact with the DNA
• 2 additional Zn ions stabilized protein fold
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The Leucine Zipper Motif
• First found in C/EBP, a DNA-binding protein in rat liver nuclei • Present in nearly all organisms
• Characteristic features: a 28-residue sequence with Leu every 7th position and a "basic region”
• An amphipathic alpha helix and a coiled-coil dimer (hydrophobic core)
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Helical wheel from C/EBP(CAAT enhancer binding protein)
• Structure of α-helix makes every 7th residue lie on one side– all the Leu (hydrophobic
residues line up)
• When two helices like this coil together the Leu stripes will form a hydrophobic core
Amphipathic helix
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bZIP Protein
• Zipper helps for dimer• Basic regions interact with DNA
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Leucine zipper (bZIP, GCN4)
• Zipper helps for dimer
• Basic regions interact with DNA
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Many proteins recognize a DNA sequence via alpha-helices positioned in the major groove of the DNA
but
there are also very different DNA binding proteins that do not fit into the simple classification of binding motifs
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TATA box binding protein at the promoter