Eukaryotic Transcription Factor TFIIIA - A Zinc Finger Protein

The structural details presented in this tutorial about Zinc finger DNA binding proteins are based on:

• Lehninger, et al. "The principals of biochemistry, 2nd edition"
• Alberts, et al. "Molecular biology of the cell, 3rd edition"
• Pabo, C., Sauer, R., (1992) "Transcription factors: Structural families and principles of DNA recognition", Annual Rev. Biochem. 61:1053-95.

The tutorial was originally authored  as Rasmol scripts by Sean O'Hearn for BMMB 597C.

  Introduction

• Zinc fingers represent an important class of nucleic acid regulatory binding proteins.
  • There are several different varieties, all linked by the utilization of zinc ions to add a structural element to the binding component.
  • The 'fingers' refer only to their appearance in schematic drawings dating from their initial discovery.
    

   

• Although primarily classified as a DNA binding protein, zinc fingers have been found to regulate ribonucleic acids, particularly in the case of retroviruses.
  

   

• The zinc finger in this demonstration is TFIIIa, a eukaryotic DNA transcription factor from Xenopus laevis, an African frog.
  • It has three zinc atoms tetrahedrally coordinated by two cysteine and two histidine side chains.
  • The Brookhaven IDcode for this protein is 1TF3.

   

• One characteristic of this motif is that it recognizes G-rich DNA.
  • <> color the guanosine in this DNA orange.
  • <> add the protein in cartoon form to the complex

  The protein

• <> This particular protein is made up of three alpha helixes (in pink) and three sets of antiparallel beta sheets (yellow).

   

• <> It also binds three zinc atoms
  • <> This is a close-up of one of the zinc atoms, illustrating the coordination of the metal ion.
    • A common characteristic of this motif is the Cys-X(2 or 4)-Cys-X12-His-X2-His amino acid sequence. This keeps the zinc nested between a helix and the  beta sheets. This coordination is very important for proper wraping and binding configuration of the protein.
    • See the cysteines<>
    • Cysteine, in the beta sheets, will use sulfur as a ligand to coordinate the zinc. See the sulfur. <>
  • <> Histidine, which contains an imizadole ring, coordinates the zinc from the helix. It is the only amino acid with a pKa near neutrality. As a result, histidine acts as a Lewis acid for the zinc ion, fixing the coordination.
    • Highlight the nitrogens on the histidine ring. One of these nitrogens carries a charge at physiological pH that acts as a ligand for the zinc. <>
  • Survey the coordination.
    <>Rotate left to right
    <>Rotate right to left
    <>Rotate up and down

   

• The zinc adds a structural element to this protein
  • it is tetrahedrally coordinated with the sulfur from the cysteine and the charge on the histidine rings
  • the protein does not fold if these amino acids are not in their proper place.
  • these sites have become targets of mutagenic research in the fight against retroviruses.

   

• <> A particular advantage of this motif is that the strength and specificity of the DNA-protein interaction could be adjusted during evolution by changes in the number of these zinc finger repeats.

  The Protein / DNA contacts

• <> The protein nestles in the major groove of the B-DNA.
  • Each finger has a similar way of docking against the DNA by making base contact with a three-base-pair subunit.
    • The second beta sheet on each complex makes contact against the sugar phosphate backbone.
    • <> In this next view, the individual strands of DNA are color coded.
    • <> Changing the backbone of the DNA to a wire allows better viewing of the placement and docking of TFIIIa to the DNA

   

• The zinc finger motif interacts with the DNA through simple repeats of argenine and histidine along the alpha helixes.
  • <>  The zinc itself makes no covert contact with the nucleic acid.
  • Arg and His are both positively charged amino acids that are attracted to the negatively charged DNA.
    <>Argenines <>Histidines
  • Histidine also interacts with the DNA from the helixes.
  • It is the positively charged side chains of these amino acids that extend into the major groove of the DNA, interacting with individual nucleotides.
    Rotate vertically. <>

   

• Zinc fingers are characteristically found on guanosine-rich strands of DNA.
  • <> It is these three-base-pair repeats of guanosine that are the target of these positively charged side chains.

Review

• Unlike other DNA binding motifs, the periodicity of the zinc finger protein structure has a simple relationship to the periodicity of the double-helical DNA.
  • <> Load the protein (cartoon)
  • In the zinc finger DNA binding motif, it is usually the sixth amino acid of the helix (Arg) that interacts with the first guanosine of the three-base-pair subsite.
    • <> Add argenine side chains (dark blue wireframe)
    • <> Add guanosines (gold spacefill)
    • <> zoom in (Rasmol zoom 300)
  • Histidine from the helix also interacts with the DNA
    • <> Add histidine backbone (cyan cartoon)
    • <> Add histidine side chains (cyan wireframe)
    • <> Rotate
    • <> Identify nitrogens of histidine side chain (red wireframe)
    • <> Change guanosines to wireframe and rotate
  • <> Here is a cleaner representation of the simple three guanosine base pair relationship with the positive charged side chains of the arginine and histidine.

   

• The zinc finger motif is common to prokaryotes and eukaryotes. Although several different variety exist, the common characteristics are:
  1. Zinc ions used for structural configuration of the quartenary form of the protein complex.
  2. A typical amino acid sequence of Cys3-Cys6-His19-His23 used to coordinate the zinc ion.
  3. The perodicity of positively charged side chains of Arg interacting with three-base-pair guanosine.
  4. Structural repeats of an alpha helix with beta sheets.