Lec 2

• Common protein DNA binding domains


  • Helix-turn-helix
  • homo/hetero-dimerization
  • 2 types of Zn finger motifs
  • Leucine Zipper
  • Helix-loop-helix
• Step#1 Transcriptional controls

  Regulate the frequency and timing of a gene's transcription to control cellular and biological processes and prevent the production of unnecessary intermediates by the cell
• Transcription is controlled by:

  
  

  a regulatory region of DNA (cis-elements) near the transcription start site called the promoter
• What are the 2 fundamental components needed for a cell to transcribe its genes?

  • Short stretches of DNA (~5-10 nt pairs) of defined sequence (cis-regulatory sequences)
  • Gene regulatory proteins that bind to these sequences (transcription factors (proteins))
• Transcription factors contain structural motifs that can 'read' specific DNA sequences


  • Proteins generally make a large number of contacts with the DNA (hydrogen bonds, ionic bonds, + charged amino acids that interact with negatively charged phosphates on DNA, and hydrophobic interactions)
  • Individual contacts may be weak but, the combination of contacts add together to ensure the interaction is highly specific and very strong
• What are the structures that bind to the major groove of DNA?

  α helices or β sheets are the secondary protein that bind
• Transcription factors contain structural motifs that can ‘read’ SPECIFIC DNA sequences

  • This specificity comes from unique hydrogen bonds between the protein side chain and particular DNA bases located in the major groove of the DNA double helix.
• What does Asn residue of a DBTF contact?

  an A in the major grove.
• What do TFs usually interact with?

  DNA bases but some a.a. can also interact with phosphate backbone
• An example of H-bonding of a common protein side chain in DNA binding transcription factors with particular DNA bases

  10 to 20 of these individual amino acid: DNA base interactions are needed for a transcription factor to recognize its particular DNA element
• The DNA binding portion of transcriptional regulators makes a series of contacts with the DNA, major groove interactions are most important!
• 10 to 20 of these individual amino acid: DNA base interactions needed for a transcription factor to recognize its particular DNA element
• Individual contacts may be weak. This DBD in this regulatory protein makes 13 contacts with the cis-regulatory element of its target gene.
• 20 contacts are the most typical.
• The cis-regulatory sequence of a gene


  DBD of a transcriptional regulator
• Examples of different DNA binding motifs
• Transcription factors may contain one or another of a small set of DNA-binding structural motifs.
• These motifs generally use either α helices or β sheets to bind to the major groove of DNA.
• The amino acid side chains that extend from these protein motifs make the specific contacts with the DNA.
• Helix-turn-helix is the most common DNA-binding motif

  • Constructed from 2 alpha helices connected by a short AA chain "turn"
  • Two helices are held at a fixed angle
  • C-terminal "recognition helix" fits into the major groove; N-term helix is more variable and helps position the recognition helix
• Examples of helix-turn-helix DNA-binding proteins
• These proteins vary enormously outside the helix-turn-helix region
• Each of the different proteins in this family can present their helix-turn-helix motif to the DNA in a unique way, increasing the number of DNA sequences recognized by this motif
• DNA-binding Zinc Finger Proteins

  • These DNA binding proteins add one or more zinc atoms as structural components ("zinc-coordinated DNA-binding motifs")
  • This protein consists of an α helix and β sheet held together by zinc
  • Usually found in clusters with alpha helix, as shown here, contacting the DNA
• Zinc Finger Proteins often have a cluster of zinc fingers
• These are arranged so that the α helix of each contacts the major groove of DNA, forming a nearly continuous stretch of α helices along the groove


  • This structure gives strength and specificity to the DNA-protein interaction
  • Each Zn finger contacts a subset of bases (2 in this example)
• β sheets can also recognize DNA

  • Information on the surface of the major groove is read by a two-stranded β sheet
  • This motif can be used to recognize many different DNA sequences
  • The exact DNA sequence recognized depends on the sequence of AA's that make up the sheet
• The leucine zipper motif mediates both DNA binding and protein dimerization


  • Leucine zipper motif consists of two α helices, one from each monomer, joined together to form a short coiled-coil
  • Just beyond the dimerization interface, the helices separate to form a Y-shaped structure contacting opposite sides of the major groove
  • The dimer "grips" the DNA
• Hetero-dimerization expands the repertoire of DNA sequences recognized by gene regulatory proteins


  • Many gene regulatory proteins, including leucine zippers, can associate with non-identical partners, forming heterodimers composed to different subunits
  • This greatly expands the repertoire of DNA-binding specificities that these proteins can display
• Helix-loop-helix motif also mediates both DNA binding and dimerization

  • Related to the leucine zipper
  • HLH motif- short α helix connected by a loop to a second, longer α helix
  • Loop flexibility allows one helix to fold back and pack against the other
  • Bind to DNA as a dimer
• Cis-regulatory sequences
• Transcriptional regulators "recognize" closely related sequences.
• Transcription regulators and cis-regulatory sequences

  • A) "logo" (motif) form depiction of a cis-regulatory sequence. 6-8 nt pairs for each monomer.
  • B) Proteins can recognize a collection of related sequences; "preferred" nucleotide is largest
  • C) Many transcription factors bind DNA as homo- or heterodimers
• This one "logo" by itself could occur every ~ 1000 nts, while an exact 6-nt sequence may occur every ~4,000 nts.
• Exact 12 nt sequences occur less frequently.
• Dimerization of transcription regulators increases their specificity and affinity for DNA.

  Heterodimerization further increases distinct binding sites and specificity.
• Distinct DNA-binding structural motifs in TF DBDs bind to cis-regulatory DNA sequences
• Transcription regulators bind cooperatively to DNA

  • Non cooperative (some cases) binding by a stable heterodimer. Binding curve has a standard exponential shape. More dimers, more binding.
  • Cooperative binding (many cases) Predominantly monomers in solution. Weak affinity for each other. Once binding occurs, it increases exponentially resulting in a sigmoidal shape in curve with sharp inflection point
  • Cis-regulatory sequence is either empty or full, rarely in between (Chapter 8; math behind this)
• In-class problems
• 1. Sequence specific DNA binding is accomplished by amino acids in proteins via interaction with the phosphate backbone of DNA. F