Protein-Nucleic Acid Interactions: General Principles

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Protein-Nucleic Acid Interactions General
Principles
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Role Structural Example Histones chromosomal
proteins Function DNA packaged into chromosomes
Role Regulatory Example Transcription
factors Function Gene Regulation
Role Enzymatic Example Polymerases,
Restriction Endonucleases Function Replication
Transcription
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Nucleic Acid Structure
Stabilizing forces hydrogen bonding van der Waals
attractions Hydrophobic interactions
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• A.T Watson-Crick A.A N1-amino symmetric
• G.C Watson-Crick A.C Reverse Wobble
• A.U Watson-Crick A.A N7-amino symmetric
• G.U Wobble G.G N1-carbonyl symmetric
• A.U Reverse Hoogsteen G.G N3-amino symmetric
• A.C Reverse Hoogsteen G.G N1-carbonyl,N7-amino
• Sheared G.A G.A N7-N1 amino-carbonyl
• G.A imino A.G N3-amino,amino-N1
• A.A.N7-amino C.C N3-amino symmetric
• G.G.N7-imino U.U 4-carbonyl-imino symmetric
• U.U imino-carbonyl U.U 2-carbonyl-imino
  symmetric
• U.C 4-carbonyl-amino U.C 2-carbonyl-amino
• A.U Reverse Watson-Crick
• G.C Reverse Watson-Crick
• G.U Reverse Wobble
• G.C N3-amino,amino-N3
• A.U Hoogsteen

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Local DNA structure
DNA is not a straight tube
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DNA binding proteins see the edges of the
basepairs in the major or minor groove
Major groove
Minor groove
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Structure of Glucocorticoid receptor
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What is it that these proteins interact
with Hydrogen bond donors Hydrogen bond
acceptors Hydrophobic residues
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These are the edge patterns a DNA binding protein
would see Notice that in the major groove,
every base pair has a unique pattern, wherease
the minor groove only has two distinct
patterns. The major groove is therefore more
informative than the minor groove
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• DNA binding Motif in protein molecules
• Helix-turn-helix
• Zn fingers (steroid receptor type)
• Bzip (leucine zipper)
• Parallel alpha helices
• Anti-parallel beta strands

Conformational Changes
Unbound conformation bound
conformation
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Zinc-finger motif

• Present in proteins that bind nucleic acids
• Zn2 ion is held between a pair of ? strands and
  ? helix

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Zn-Finger motif
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Leucine Zipper Motif 1YSA - Gcn4 Complex With
Ap-1 Dna from Saccharomyces cerevisiae.
Top
Front
Side
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Helix Turn Helix Motif 3CRO 434 Cro Protein
Complex With DNA Containing Operator OR1 from
Bacteriophage 434
Top
Front
Side
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What parts of the protein are involved in DNA
recognition
Mutations in helix 2 prevent DNA binding, which
can be suppressed by mutations in the DNA
sequence of the operator
Swapping helix 2 between two different repressors
also swapped the operator to which the proteins
bind
This shows that helix 2 is involved in DNA
recognition
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Explains why the same protein can bind to
different, yet related sequences with different
affinities. We saw this for the LysR type
proteins!
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Many DNA binding proteins are dimers, e.g, the
LysR type proteins and CAP
This means that there are two helix-turn-helix
motifs per dimeric protein
These will interact with two adjacent major
grooves, ie 10 bp apart
(CbbR binding site, lecture 5)
The DNA recognition site is therefore frequently
an inverted repeat
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The 434 cro molecule contains 71 amino acid
residues that show 48 sequence identity to the
69 residues that form the N-terminal DNA-binding
of 434 repressor. It is not surprising,
therefore, that their three-dimensional
structures are very similar. Like its lambda
counterpart, the subunit structure of the
DNA-binding domain of 434 repressor, as well as
that of 434 cro, consist of a cluster of four ?
helices, with helices 2 and 3 forming the
helix-turn-helix motif.
The two HTH motifs are at either end of the dimer
and contribute the main protein-DNA interactions,
while protein-protein interactions at the
C-terminal part of the chains hold the two
subunits together in the complexes. Both 434 cro
and repressor fragment are monomers in solution
even at high protein concentrations, whereas they
form dimers when they are bound to DNA.
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The protein-DNA interactions have been analyzed
in detail. Residues of the recognition ? helix
project their side chains into the major groove
and interact with the edges of the DNA base pairs
on the floor of the groove. Gln(Q)28 forms two
hydrogen bonds to N6 and N7 of Ade1 in the base
pair 1(T14-A1), and Gln29 forms hydrogen bonds
both to O6 and N7 of G13 in base pair 2
(G13-C2). At base pair 3 (T12-A3) no hydrogen
bonding to the protein occurs and direct contacts
are all hydrophobic The methyl groups of the
side chains of Thr27 and Gln29 form a hydrophobic
pocket to receive the methyl group of T12.
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The first three base pairs in all six operator
regions recognized by phage 434 repressor are
identical. This means that interactions between
these three base pairs and the two glutamine
residues (28 and 29) cannot contribute to the
discrimination between the six binding sites in
the DNA rather, these interactions provide a
general recognition site for operator regions.
This simple paattern of hydrogen bonds and
hydrophobic interactions therefore accounts for
the specificity of phage 434 cro and repressor
protein for 434 operator regions.
Note that when glutamines 28 and 29 are replaced
by any other amino acid, the mutant phages are no
longer viable.
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It is apparent from crystal structures of these
protein-DNA complexes that the differential
affinities of 434 repressor and cro for the
different operator regions are not determined by
sequence-specific interactions between amino acid
side chains of the recognition helix and base
pairs in the major groove of DNA. Instead, they
seem to be determined mainly by the ability of
the DNA to undergo specific structural changes so
that complementary surfaces are formed between
the proteins and the DNA. Nonspecific
interactions between the DNA sugar-phosphate
backbone and the proteins are one important
factor in establishing such structural changes.
In all complexes studies the protein subunit is
anchored across the major groove with extensive
contacts along two segment of the sugar-phosphate
backbone, one to either side of the groove.
Hydrogen bonds between the DNA phosphate groups
and peptide backbone NH groups are remarkably
prevalent in these contacts.
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One of these interaction regions involves the
loop after the recognition helix, where three
main-chain NH groups form hydrogen bonds with
phosphatess 9 and 10. All residues in this
loop, which are outside the HTH motif, contribute
to the surface complementarity between the
protein and the sugar-phosspahte surfaces of
nucleotides 9 and 10.
These and other nonspecific interactions, which
stabilize the appropriate DNA conformation,
involve a large number of residues that are
distributed along most of the polypeptide chain.
Thus the  unit  that is responsable for
differential binding to different operator DNA
regions is really an entire binding domain, and
nearly all the protein-DNA contacts contribute to
this specificity.
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Tata-box binding protein PDBcode 1cdw R 1.9 Å
R factor 0.189
The ternary complexes DNA/TBP/TFIIA and
DNA/TBP/TFIIB are now available. The
superposition gives the following multicomplex
structure.
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Histone octamer
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Histone tails between DNA gyres
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Two halves of DNA wrapped around an octamer
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RNA polymerase II (?4/7) Crystal structure at
2.8Å resolution
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RNA polymerase II (?4/7) Crystal structure at
2.8Å resolution



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Four crystal structures of RNA polymerase II
transcribing complexes
4. Post-translocation
3. Pre-translocation