Chapter 9 DNA recognition by eukaryotic transcription factors

Chapter 9 DNA recognition by eukaryotic transcription factors

TRANSCRIPTION 101

Eukaryotic RNA polymerases RNA polymerase RNA polymerase I RNA polymerase II RNA polymerase III RNA polymerase IV Function rrna mrna, snrna, mirna trna 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

A β sheet in TBP forms the DNA-binding site 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

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: hydrophobic Side chains from the central β strands Phosphate sugar backbone Minor groove DNA sequence-specific contacts No 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

Nitrogenous bases Pyrimidines Cytosine (C) Thymine (T, in DNA) Uracil (U, in RNA) Purines Adenine (A) Guanine (G)

TFIIA and TFIIB bind to both TBP and DNA

Eukaryotic helix-turn-helix motif HOMEODOMAIN PROTEINS

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

Helix-turn-helix motif of homeodomain is similar to prokaryotic counterparts with deviations - Monomeric homeodomain proteins bind specifically to DNA fragments containing the sequence 5 -A-T-T-A-3 with a K d ~1 nm. - Nonspecifically to different sequences with about 100 times lower affinity

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

Another eukaryotic helix-turn-helix motif POU DOMAIN

POU Domain POU: Pit-1, Oct-1&2, Unc-86 Pituitary-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 regions bind to DNA by two tandemly oriented helix-turn-helix motifs POU s POU H

Sequence-specific contacts between DNA and the POU region

Flexibility of POU domain [Ryan and Rosenfeld, Genes Dev. (1997)]

Tumorigenesis and DNA recognition P53

[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

Understanding tumorigenic mutations (p53) L330H [Joerger and Fersht, Oncogene (2007)]

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