Lambda Repressor cI / DNA Interactions

The structural details presented in this tutorial are based on the review by Pabo and Sauer (1992), Annu. Rev. Biochem. 61:1053-1095.

• The lambda repressor cI is an example of the helix-turn-helix family of DNA binding proteins. Many prokaryotic transcription factors belong to this family, including

  CAP	TrpR	LacR	GalR	Tet(Tn10)R	s54-dependent activators such as NtrC, NifA, XylR, DctD
  434 Rep	434 Cro	l Cro	l Rep	Fis

  
  

• The HTH "domain" is part of a larger protein; by itself, it does not fold into a separate, stable domain.
  • For example, in FIS, an extra helix is present which provides a dimerization domain;
  • this same motif appears in NtrC's DNA binding domain, but is not present in other s⁵⁴-dependent activators.
• Recognition of DNA binding sites by proteins containing an HTH motif includes contacts made by
  • the so-called recognition helix, the second one in the motif,
  • and also contacts made by the first helix and other surrounding protein parts.
• The ability of the DNA site to assume specific conformations favorable to binding also contributes to specificity.

Overall Structure

• The HTH motif is composed of two helixes separated by a turn
• In the figure, helix 1 (red) sits in the major groove,
• while helix 2 (blue) positions it, and makes makes contact with the backbone of the DNA.
• Note that the structure is actually part of a larger unit, and does not fold into a stable domain by itself.
• It may help you to understand structure by viewing it from different perspectives. You can use your mouse (click and drag) to rotate the present view, change the presentation using MDL Chime commands, or simply by pressing this button to run a script that makes the view shown rotate. <>

The atoms that make contact.

<> The DNA template as white Van der Waal spheres

• <> DNA backbone atoms within 1200 rasmol units of protein as red spheres.
• <> DNA non backbone atoms within 1200 rasmol units of the protein as blue spheres
• <> protein atoms within 1200 rasmol units of the DNA backbone atoms as pink spheres
• <> protein atoms within 1200 rasmol units of the DNA non-backbone atoms as cyan spheres.

Contacts made by the"Recognition Helix" - Helix 2

The recognition helix residues Gln44, Gln33, Ser45, Gly46, Gly48, and Asn52 make specific contacts.

• Two atoms of Gln44 make hydrogen bonds to nitrogens of A24 of the DNA.
  • <> Gln44 (yellow) opposite A24.
  • <> OE1 (red) and NE2 (blue) of Gln44, and N6 (orange) and N7 (cyan) of the adenine base.
  • The image is now centered on OE1. Use the mouse to rotate about this atom to inspect these hydrogen bonds.

• Gln33 contacts a phosphodiester oxygen and also hydrogen bonds to the Gln44 side chain.
  • <> Gln33 (red) and Gln44 (yellow).
  • <> NE2 and OE1 of Gln33 in Van der Waals spheres, and atoms O1P of the DNA backbone plus NE2 of Gln44.
  • The view is now centered on OE1 of Gln33.

• Atom OG of Ser45 makes a single hydrogen bond to N7 of G16.
  • <> Ser45 (yellow) opposite G16.
  • <> NE2 and OE1 of Gln33 in Van der Waals spheres, and atoms O1P of the DNA backbone plus NE2 of Gln44.
  • The view is now centered on OG of Ser45.

• Gly46 and Gly48 make hydrophobic contacts with methyl groups of T15 and T25.
  • <> Gly46 (yellow) opposite T15.
  • <> Gly46 CA carbon and T25 methyl group.
  • The view is now centered on CA of Gly46.
  • <> Gly48 (yellow) opposite T25.
  • <> Gly48 CA carbon and T25 methyl group.
  • The view is now centered on CA of Gly48.

• Asn52 makes a phosphodiester oxygen contact to A2.
  • <> Asn52 (yellow) opposite A2.
  • <> ND2 of Asn52 and O1P of A2.
  • The view is now centered on OG of Ser45.

• <> Review these contacts of the recognition helix binding to the site. One of the half sites shows the contacts in Van der Waals sphere, and the other simply shows the amino acids that make contact in wireframe that is colored red.

Residues outside of the "Recognition Helix" make contacts

• Lys19 and Tyr22 illustrate that residues outside of the so-called recognition helix are important for specific binding.
  • <> Lys19 and Tyr22
• Lys4 and Asn55 cooperate to make additional hydrogen bonds to a G in the major groove, and additional contacts to backbone phosphates are important.
  • <> Lys4 and Asn55
• Finally, several backbone contacts are made by residues additional residues.
  • <> These include Lys26, Met42, Gly43, Asn58, and Asn61.

Summary

The phage lambda repressor serves as an example of the Helix-turn-Helix DNA binding motif of prokaryotic transcription factors. The second helix makes sequence specific contacts, but so do residues of helix 1, and of other parts of the protein. These contacts include hydrogen bonds between lysine, arginine, short polar side chains, or even by -NH groups from the polypeptide backbone. There are also some hydrophobic interactions between methyl groups of thymine and glycine residues. Sometimes bridges occur between three residues which can be imagined to anchor the overall structure.

<> I leave you with a model with all contacts shown, with red for backbone DNA atoms, pink for protein in contact with DNA backbone, blue for non-backbone DNA atoms, and cyan for protein in contact with these.