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Protein-DNA Interactions
Site-specific
Blackburn & Gait, p. 400-415, 418-421
Neidle, chapter
Understand the basics behind HTH motif
• important AAs, how protein recognizes DNA,
dimerization, important contacts
• examples: CAP, cro repressor, etc.
Understand the basics behind Homeodomain motif
• important AAs, how protein recognizes DNA,
monomer, important contacts
• examples: Drosophila proteins, yeast MATa2
Understand the basics behind Zinc finger motif
• important AAs, how protein recognizes DNA,
3 types, important contacts
• examples: Zif268, glucocorticoid receptor, GAL4
Understand the basics behind leucine zipper motif
• important AAs, how protein recognizes DNA,
dimerization, important contacts
• examples: GCN4, jun, fos
Understand the basics behind TBP binding to DNA
• important AAs, how protein recognizes DNA,
saddle shaped structure, important contacts
Understand the basics behind RNP motif
• loops of protein bind RNA
• U1A protein binds toU1 snRNA
Understand the basics HIV TAR RNA binding by Tat
• important AAs, how protein recognizes DNA,
important contacts
Protein-DNA Interactions
Site-specific
For cell to function proteins must distinguish 1 nucleic acid
sequence from another very accurately
• tRNA synthetase must charge only its cognate tRNA
• transcriptional activators and repressors must turn on specific
genes
We understand protein-nucleic acid interactions mostly from
crystal structure and NMR data
Protein-DNA Interactions
Site-specific
Helix-turn-helix (transcriptional regulators)
• no stable structure by itself, needs surrounding protein sequence
• first sequence-specific DNA binding protein structures solved
were from proks - E.Coli CAP (catabolite activator protein) & cro
repressor from phage l
• both have HTH - most common sequence-specific DNA binding
motif
• Other examples: 434 cro, lambda repressor, 434 repressor, trp
repressor
• sometimes in euks - homeodomain motif (talk about later)
Protein-DNA Interactions
Site-specific
Helix-turn-helix (transcriptional regulators)
Structure well-established, ~20 amino acids
Pair of helices that stack to form a V-shape (60˚ angle)
Usually first helix positions the second (recognition helix) so that
it projects into MAJOR groove and recognizes specific sequence
Protein-DNA Interactions
Site-specific
Helix-turn-helix (transcriptional regulators)
Structure well-established, ~20 amino acids
6 AAs out of 20 in motif help maintain correct angle
Position -9 at turn between 2 helices usually Gly
Positions -4, -8, -10, -15 usually hydrophobic
Position -5 usually small (Ala or Gly)
Motif always occurs as part of a larger structure that differs from
protein to protein
Protein-DNA Interactions
Site-specific
Helix-turn-helix (transcriptional regulators)
Functions as dimer
DNA sequence has twofold symmetry
Recognition helix is a misnomer - both helices contact DNA
Each monomer recognizes half-site
Helix #2 is above MAJOR groove but its N-term is in contact
with phosphate backbone
Protein-DNA Interactions
Site-specific
Helix-turn-helix (transcriptional regulators)
Repressor forms H-bonds to 4 phosphates per monomer,
clamping helix #3 (recognition helix) in MAJOR groove
Repressor uses backbone amides and side chain groups
Protein-DNA Interactions
Site-specific
Helix-turn-helix (transcriptional regulators)
CAP - 90˚ bend in DNA, little change to protein
Ethyl-phos
No pro bind
Phos sensitive
To DNase I
Protein-DNA Interactions
Site-specific
Helix-turn-helix (transcriptional regulators)
CAP - two kinks ~43˚ each
Bases roll and unstack base pair at TG
Protein-DNA Interactions
Site-specific
Helix-turn-helix (transcriptional regulators)
CAP - Glu181 critical to form kink
Electrostatics important - lots of Lys and Arg
Protein-DNA Interactions
Site-specific
Homeodomain - Eukaryotic motif
Similar to HTH? NO can fold by itself
Binds euk. asymmetric homeobox sequence as monomer
~60 AA module found in: Dros. Antennapedia, Dros. Engrailed,
yeast MATa2
Protein-DNA Interactions
Site-specific
Homeodomain - Eukaryotic motif
Bind DNA by inserting long 3rd helix (recognition helix) into
MAJOR groove and N-term arm into adjacent MINOR groove
Protein-DNA Interactions
Site-specific
Homeodomain - Eukaryotic motif
IMPORTANT - Asn51 makes two H bonds to A in MAJOR
groove
Additional links to phosphate backbone
AA 47, 50 and 54 help discriminate one homeobox from another
Protein-DNA Interactions
Site-specific
Zinc finger - Eukaryotic motif
Most common euk. gene regulatory proteins have Zn fingers
Zinc coordinated to Cys or His of DNA binding domain
1st discovered was from Xenopus - TFIIIA
Three types:
Cys2-His2
Cys4
GAL4 dinuclear cluster
All use a-helices in MAJOR groove
Structural data from crystallography and NMR
~30 AA domain binds Zn and folds properly
Protein-DNA Interactions
Site-specific
Zinc finger - Eukaryotic motif
Cys2-His2
3 tandemly repeated Zinc fingers
1 Zinc finger
Protein-DNA Interactions
Site-specific
Zinc finger - Eukaryotic motif
Cys2-His2
Zn staples a-helix & b-sheet together as well as forms a phobic core
Zif268 contacts to G-rich DNA strand by Arg/His
Protein-DNA Interactions
Site-specific
Zinc finger - Eukaryotic motif
Cys4 nuclear receptors
Two a-helix loop motifs bind as dimer to 2-fold symmetrical sequence
GRE (glucocorticoid response element) in DNA is bound by dimer of
glucocorticoid receptor
GRE is made of 2 half-sites
5’-AGAACA XXX TGTTCT-3’ (has to be a 3-nt spacer)
Protein-DNA Interactions
Site-specific
Zinc finger - Eukaryotic motif
Cys4 nuclear receptors
GRE (glucocorticoid response element) in DNA is bound by dimer of
glucocorticoid receptor
Protein-DNA Interactions
Site-specific
Zinc finger - Eukaryotic motif
GAL4
• Has 3 subunits - Zn cluster, linker, dimerization region
• 2 Zn ions coordinated by 6 Cys
• Monomeric in solution but dimerizes upon binding 17 bp
symmetrical DNA sequence with specific CCG triplets at ends
Protein-DNA Interactions
Site-specific
Leucine Zipper (bZIP) - Eukaryotic motif
• Found in certain euk regulatory DNA-binding proteins
• Ex: yeast transcriptional regulatory protein GCN4 & AP1
(oncoproteins jun and fos)
• Leucine zipper does not bind DNA but dimerizes proteins so they can
bind DNA
• Leu zipper is an amphipathic a-helix where phobics (Leu) face one
side and charged AAs the other
• bZIP <100 AAs, three domains (N-term regulatory, dimerization
leucine zipper, basic DNA binding)
• Leucine zipper - a-helix with Leu every 7 AAs, since a-helix has 3.6
AA/turn of helix all Leucines on the same face
Protein-DNA Interactions
Site-specific
Leucine Zipper (bZIP) - Eukaryotic motif
• Leucine zipper - a-helix with Leu every 7 AAs, since a-helix has 3.6
AA/turn of helix all Leucines on the same face
• two basic ends form a-helices and sit in MAJOR groove
• Dimerization allows protein to bind DNA in scissors-grip fashion
(Y-shape)
• 2-fold symmetry
Protein-DNA Interactions
Site-specific
Basic Helix-Loop-Helix Zipper (bHLHZ) - Eukaryotic motif
Protein-DNA Interactions
Site-specific
TATA Box binding protein - Eukaryotic motif
To initiate transcription all three RNA polymerases require TATA box
binding protein (TBP) which binds to MINOR groove of DNA and
recognizes TATA sequence
TBP  5-stranded all antiparallel b-sheet & domains connected by
7-AA linker
Saddle-shaped structure
Regulation of iron metabolism (eukaryotes)
•
The level of free iron is highly regulated in eukaryotes
•
Two opposing protein activities are that of the
transferrin receptor which transports iron into cells, and
ferritin which stores iron
•
The expression of each of these proteins is oppositely
regulated at the translational level by the same ironsensitive factor
•
The iron-response
element (IRE) is an
RNA sequence
specifically recognized
and bound by the
IRE-binding protein
(IRE-BP)
•
IRE-BP binding to
iron or the IRE is
mutually exclusive
•
IRE-BP binding to
ferritin mRNA
inhibits translation
while IRE-BP binding
to the transferrin
receptor mRNA
stabilizes the mRNA
and promotes
translation
IRE-BP
The iron response element
ferritin mRNA
transferrin receptor mRNA
Protein-DNA Interactions
Site-specific
RNA binding proteins
RNP motif/domain ~90 AA sequence
Example: U1A protein binds to U1 snRNA
Tat-TAR in HIV
Activity of Tat
•
Bulge loop in nascent HIV transcript is recognized by
regulatory protein
•
Protein is Tat, trans-activator protein
•
Binding site is TAR, trans-activation response element
•
Tat-TAR interaction is required for HIV transcription
•
Tat stimulates full
length viral RNA
transcription
•
Without Tat,
transcripts are short
•
With Tat,
transcripts are full
length
Tat Binding Site
•
Tat protein binds to trinucleotide bulge
in TAR RNA
•
Arginine rich basic region of Tat binds
TAR
•
Causes a complete rearrangement in
TAR conformation
TAR RNA
Tat Protein
Protein-DNA Interactions
Site-specific
RNA binding proteins
HIV TAR - Tat interaction
Tat - Trans-activating protein (86 AA)
TAR - Trans-activating RNA
Protein-DNA Interactions
Site-specific
RNA binding proteins
HIV TAR - Tat interaction
TAR Structures
Without Tat
With Tat
•
Bulge closes upon binding
•
Other factors also bind
•
Changes the processivity of RNA pol
•
Induced fit binding
Activity of Tat-TAR
•
•
•
•
Tat binding recruits CyclinT-cdk9 to TAR
Also recruits TFIIH to TAR
Both phosphorylate the CTD of RNA pol II
Improves the elongation efficiency of pol II
Protein-DNA Interactions
Site-specific
RNA binding proteins
HIV TAR - Tat interaction