Chapter 9DNA recognition by eukaryotic
transcription factors
TRANSCRIPTION 101
Eukaryotic RNA polymerases
RNA polymerase Function
RNA polymerase I rRNA
RNA polymerase II mRNA, snRNA, miRNA
RNA polymerase III tRNA
RNA polymerase IV siRNA (plants)
snRNA (small nuclear RNA): small RNA found inside the nucleus functioning in RNA splicing and telomere maintenance
miRNA (microRNA): single-stranded RNA with 21-23 nucleotides functioning in regulating gene expression
siRNA (small interfering RNA): double-stranded RNA with 20-25 nucleotides functioning in RNA intereference
Transcription is activated by protein-protein interactions
Eukaryotic pre-initiation complex
TATA-BOX BINDING PROTEIN (TBP)
The three-dimensional structures of TBP-TATA box complexes are known
“Saddle-shaped molecule”:C-terminal 180 residues form two structurally similar motifs (88 residues), anti-parallel β sheet of 5 strands
→ unlike prokaryotic DNA-binding proteins
A β sheet in TBP forms the DNA-binding site
TBP binds in the minor groove and induces large structural changes in DNA
110 °
The interaction are between TBP and the TATA box is mainly hydrophobic
Interaction area: hydrophobicSide chains from the central β strandsPhosphate sugar backboneMinor groove
DNA sequence-specific contactsNo G-C pair allowed
DNA sequence-specific hydrogen bonds:At the center of the minor groove
• Minor groove recognition• Easier bendability of A:T pairs
Purines
Guanine (G)Adenine (A)
Cytosine (C) Thymine (T, in DNA) Uracil (U, in RNA)
Nitrogenous basesPyrimidines
TFIIA and TFIIB bind to both TBP and DNA
HOMEODOMAIN PROTEINSEukaryotic helix-turn-helix motif
Homeodomain proteins are involved in the development of many eukaryotic organism
Monomers of homeodomain proteins bind to DNA through a helix-turn-helix motif
Helix-turn-helix motif of homeodomain is similar to prokaryotic counterparts with deviations
Monomer binds DNA Dimer binds DNA
- Monomeric homeodomain proteins bind specifically to DNA fragments containing the sequence 5’-A-T-T-A-3’ with a Kd ~1 nM.- Nonspecifically to different sequences with about 100 times lower affinity
Helix-turn-helix motif of homeodomain is similar to prokaryotic counterparts with deviations
Overall arrangement of the homeodomains bound to DNA is virtually identical (seq. identity is only 20%)
In vivo specificity of homeodomain transcription factors depends on interactions with other proteins
POU DOMAINAnother eukaryotic helix-turn-helix motif
POU: Pit-1, Oct-1&2, Unc-86Pituitary-specific Pit-1 Octamer transcription factor proteins Oct-1 and Oct-2 (octamer sequence is ATGCAAAT) neural Unc-86 transcription factor from Caenorhabditis elegans
[Ryan and Rosenfeld, Genes Dev. (1997)]
POU Domain
POUs POUH
POU regions bind to DNA by two tandemly oriented helix-turn-helix motifs
Sequence-specific contacts between DNA and the POU region
Flexibility of POU domain
[Ryan and Rosenfeld, Genes Dev. (1997)]
P53Tumorigenesis and DNA recognition
[Toledo and Wahl, Nat. Rev. Cancer (2007)]
The monomeric p53 polypeptide chain is divided in 3 domains
- The oligomerization domain forms tetramers: some mutations detected in tumor are in this domain - Leu330 → His (in the hydrophobic core in a dimer); destabilizes dimer - Gly in the turn between the β-strand and the α-helices; any mutation will cause energetically unfavorable folding
[Joerger and Fersht, Oncogene (2007)]
L330H
Understanding tumorigenic mutations (p53)
Structure of the DNA-binding domain of p53 (anti-parallel β barrel: 9 β strands)
- Protruding loops (L1,3) to both minor and major grooves - Two loops and one α helix bind to DNA- Zn stabilizes a loop conformation (2 Cys from L3 & Cys/His from L2) - There are also non-specific interactions between p53 and DNA - R280 in helix ↔ major groove (conserved DNA sequence)
Out of 21 bp, 10 bp are involved in the sequence-specific binding
Yunje Cho @POSTECH
Tumorigenic mutations occur mainly in three regions involved in DNA binding
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