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1. Beskrive de vigtigeste principper for DNA-protein interaktion og angive hvilken betydning DNA-major groove har. Beskrive helix-turn-helix motiver og zink-finger. Definere leucin-zippers og beskrive hvorledes de er involveret i protein-protein interaktion med DNA

 

Principper for DNA-protein interaktion
 Devlin, s. 387
Devlin 390-391, fig. 9.27
Stryer, s.795, fig. 28.20
Stryer, s. 867

The entire idea with DNA-protein interaction is the need of controlling the gene expression. Two things have to be considered:

There are ca. 30000 genes in the human genome, and each of them has a regulatory sequence called the transcription factor binding element. This element can be:

- a promoter - located upstream and close to the target gene
- an enhancer - located thousands of nucleotides away from the target gene

The DNA-binding proteins, known as transcription factors or regulatory proteins recognize this regulatory sequence due to non-covalent interactions between the protein and DNA, and bind to it.

The interplay between the proteins DNA-binding site and the DNA-sequence controls the gene expression at transcription level. The transcription factor usually binds the RNA polymerase that transcribes the gene.

fx.
DNA-binding proteins utilize a variety of strategies for interaction with DNA. I will take the TATA-TBP complex as an example.

The TATA box is the promoter sequence in DNA found in eukaryotes, while TBP is the TATA-box binding protein.

TBP is a saddle-shaped protein consisting of two similar domains, each composed of a curved antiparallel B-sheet with a concave surface. The TATA box of DNA binds to the concave surface of TBP. The double helix is substantially unwound to widen its minor groove, enabling it to make very extensive contact with the antiparallel B-strands on the concave side of TBP.
Immediately, outside the TATA box, classical B-DNA resumes.

TBP bound to TATA box is the heart of the initiation complex. The surface of TBP saddle provides docking sites for the binding of other components, among which RNA polymerase II.

Betydning af DNA-major groove
 Devlin, s. 40
Devlin, s. 38-39 - fig.2.15; 2.16
Stryer, s. 748, fig. 27.8

The major groove of DNA is the binding site for the different transcription factors. Its larger size makes it more accessible for interactions with proteins that recognize specific DNA sequences. 

Because each of the four bases has its own orientation with respect to the rest of the helix, each base always displays the same atoms into the grooves. These atoms then constitute an important means of sequence-specific recognition of DNA by other molecules such as proteins.

fx.
the N7 of purines is always displayed in the major groove and can serve as hydrogen-bond acceptor in interactions with donor groups of proteins

Helix-turn-helix (HTH)
 Devlin, s.354-355, fig. 8.24
 Devlin, s. 387,  fig. 9.20
Stryer, s. 874

This is the most common structural motif found in many DNA-binding proteins. It is made up of a pair of alfa-helices and a tight turn:

- the first alfa-helix -  made up of 7 amino acids, participates primarily in interactions with the DNA-backbone, as well as hydrophobic interactions with the second helix, stabilizing the structure.

- the second alfa-helix - also called  the recognition helix is made up of 9 amino acids and is positioned across the major groove, where side chain residues of the helix form specific non-covalent interactions with the base sequence of the target DNA.

Most HTH-proteins bind as dimers, each monomer domain binding at 2 adjacent turns of the major groove of DNA on the same side of the molecule. The distance between the monomers is 34Å, which corresponds in one turn of the DNA-molecule. The dimer dual-binding interaction makes a much stronger and more specific protein-DNA interaction than possible for a single monomer protein-DNA interaction. The interaction induces a distorsion in the DNA structure so that the protein is easily and better accomodated.

Zink-finger
 Devlin, s. 389, fig. 9.22
Stryer, s. 880

The name of the zink-finger domain comes from the importance of the Zn-jons it contains. The Zn-jons stabilize the structure of the domain; without them the domains unfold.

The primary structure for the motif contains 2 close cysteins separated by 12 a. a. from a second pair of Zn-liganding amino acids (most often to histidines, but sometimes they can be substituted by cysteins). The two cysteins in the first pair are separated by only two amino acids, while the histidines/cysteins in the second pair can be separated by three amino acids.

The motif contains an alfa-helix segment that can bind within the major groove at its target site in DNA and makes specific interactions with the nucleotide sequence. The alfa-helix segment is located at the end of the Zn-finger domain.

Leucine zippers
 Devlin, s. 356 - fig. 8.26
Devlin, s. 389 - fig. 9.24

Leucine zippers, also called basic-region leucine zippers, are formed from a region of alfa-helix that contains at least 4 leucines, each leucine separated by six amino acids from one another. With 3.6 residues per turn of the alfa-helix, the leucines align on the edge of the helix, with a leucine at every second turn of the helix.

Protein-protein interaction: The leucine rich helix forms a hydrophobic interaction with a second leucine helix on another protein to zipper the two together to form a dimer. The formation of the zipper is necessary in order to form a strong bond with the DNA.

The leucine zipper motif does not interact directly with DNA.

The DNA-binding region is adjacent to the zipper motif. It is a basic region, defined by the presence of arginine and lysine (that is why the protein is called basic-region leucine zippers). This basic region assumes the conformation of two alfa-helices with a small break that allows the helices to follow the major groove of the DNA.

The basic amino acids serve to stabilize the DNA-protein association through electrostatic interactions between the positively charged amino acids and the negatively charged DNA backbone.

Many regulatory proteins containing a leucine zipper are oncogenes. If the regulatory proteins are mutated or produced in a non-regulated fashion, the cell can be transformed into a cancer cell. 

 

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