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