Egr1

EGR-1 control region

| | WT1 binding site | | SRF/TCF binding site | | API binding site

▲ FIGURE 11-19 Diagram of the control region of the gene encoding EGR-1, a transcription activator. The binding sites for WT1, a eukaryotic repressor protein, do not overlap the binding sites for the activator API or the composite binding site for the activators SRF and TCF Thus repression by WT1 does not involve direct interference with binding of other proteins as in the case of bacterial repressors.

early in life. The WT1 protein binds to the control region of the gene encoding a transcription activator called EGR-1 (Figure 11-19). This gene, like many other eukaryotic genes, is subject to both repression and activation. Binding by WT1 represses transcription of the EGR-1 gene without inhibiting binding of the activators that normally stimulate expression of this gene. I

DNA-Binding Domains Can Be Classified into Numerous Structural Types

The DNA-binding domains of eukaryotic activators and re-pressors contain a variety of structural motifs that bind specific DNA sequences. The ability of DNA-binding proteins to bind to specific DNA sequences commonly results from non-covalent interactions between atoms in an a helix in the DNA-binding domain and atoms on the edges of the bases within a major groove in the DNA. Interactions with sugar-phosphate backbone atoms and, in some cases, with atoms in a DNA minor groove also contribute to binding.

The principles of specific protein-DNA interactions were first discovered during the study of bacterial repressors. Many bacterial repressors are dimeric proteins in which an a helix from each monomer inserts into a major groove in the DNA helix (Figure 11-20). This a helix is referred to as the recognition helix or sequence-reading helix because most of the amino acid side chains that contact DNA extend from this helix. The recognition helix that protrudes from the surface of bacterial repressors to enter the DNA major groove and make multiple, specific interactions with atoms in the DNA is usually supported in the protein structure in part by hydrophobic interactions with a second a helix just N-terminal to it. This structural element, which is present in many bacterial repressors, is called a helix-turn-helix motif.

Many additional motifs that can present an a helix to the major groove of DNA are found in eukaryotic transcription factors, which often are classified according to the type of DNA-binding domain they contain. Because most of these motifs have characteristic consensus amino acid sequences, newly characterized transcription factors frequently can be classified once the corresponding genes or cDNAs are cloned

| | WT1 binding site | | SRF/TCF binding site | | API binding site

▲ FIGURE 11-20 Interaction of bacteriophage 434 repressor with DNA. (a) Ribbon diagram of 434 repressor bound to its specific operator DNA. Repressor monomers are in yellow and green. The recognition helices are indicated by asterisks. A space filling model of the repressor-operator complex (b) shows how the protein interacts intimately with one side of the DNA molecule over a length of 1.5 turns. [Adapted from A. K. Aggarwal et al., 1988, Science 242:899.]

and sequenced. The genomes of higher eukaryotes encode dozens of classes of DNA-binding domains and hundreds to thousands of transcription factors. The human genome, for instance, encodes «2000 transcription factors.

Here we introduce several common classes of DNA-binding proteins whose three-dimensional structures have been determined. In all these examples and many other transcription factors, at least one a helix is inserted into a major groove of DNA. However, some transcription factors contain alternative structural motifs (e.g., p strands and loops) that interact with DNA.

Homeodomain Proteins Many eukaryotic transcription factors that function during development contain a conserved 60-residue DNA-binding motif that is similar to the helix-turn-helix motif of bacterial repressors. Called homeo-domain proteins, these transcription factors were first identified in Drosophila mutants in which one body part was transformed into another during development (Chapter 15). The conserved homeodomain sequence has also been found in vertebrate transcription factors, including those that have similar master control functions in human development.

Zinc-Finger Proteins A number of different eukaryotic proteins have regions that fold around a central Zn2+ ion, producing a compact domain from a relatively short length of the polypeptide chain. Termed a zinc finger, this structural motif was first recognized in DNA-binding domains but now is known to occur also in proteins that do not bind to DNA. Here we describe two of the several classes of zinc-finger motifs that have been identified in eukaryotic transcription factors.

The C2H2 zinc finger is the most common DNA-binding motif encoded in the human genome and the genomes of most other multicellular animals. It is also common in mul-ticellular plants, but is not the dominant type of DNA-binding domain in plants as it is in animals. This motif has a 23- to 26-residue consensus sequence containing two conserved cysteine (C) and two conserved histidine (H) residues, whose side chains bind one Zn2+ ion (see Figure 3-6b). The name "zinc finger" was coined because a two-dimensional diagram of the structure resembles a finger. When the three-dimensional structure was solved, it became clear that the binding of the Zn2+ ion by the two cysteine and two histi-dine residues folds the relatively short polypeptide sequence into a compact domain, which can insert its a helix into the major groove of DNA. Many transcription factors contain multiple C2H2 zinc fingers, which interact with successive groups of base pairs, within the major groove, as the protein wraps around the DNA double helix (Figure 11-21a).

A second type of zinc-finger structure, designated the C4 zinc finger (because it has four conserved cysteines in contact with the Zn2+), is found in «50 human transcription factors. The first members of this class were identified as specific in-tracellular high-affinity binding proteins, or "receptors," for steroid hormones, leading to the name steroid receptor superfamily. Because similar intracellular receptors for non-steroid hormones subsequently were found, these transcrip tion factors are now commonly called nuclear receptors. The characteristic feature of C4 zinc fingers is the presence of two groups of four critical cysteines, one toward each end of the 55- or 56-residue domain. Although the C4 zinc finger initially was named by analogy with the C2H2 zinc finger, the three-dimensional structures of proteins containing these DNA-binding motifs later were found to be quite distinct. A particularly important difference between the two is that C2H2 zinc-finger proteins generally contain three or more repeating finger units and bind as monomers, whereas C4 zinc-finger proteins generally contain only two finger units and generally bind to DNA as homodimers or heterodimers. Ho-modimers of C4 zinc-finger DNA-binding domains have twofold rotational symmetry (Figure 11-21b). Consequently, homodimeric nuclear receptors bind to consensus DNA sequences that are inverted repeats.

Leucine-Zipper Proteins Another structural motif present in the DNA-binding domains of a large class of transcription factors contains the hydrophobic amino acid leucine at every seventh position in the sequence. These proteins bind to DNA as dimers, and mutagenesis of the leucines showed that they were required for dimerization. Consequently, the name leucine zipper was coined to denote this structural motif.

The DNA-binding domain of the yeast GCN4 transcription factor mentioned earlier is a leucine-zipper domain. X-ray crystallographic analysis of complexes between DNA and the GCN4 DNA-binding domain has shown that the dimeric protein contains two extended a helices that "grip" the DNA molecule, much like a pair of scissors, at two ad-

► FIGURE 11-21 Interaction between DNA and proteins containing zinc fingers.

(a) GL1 is a monomeric protein that contains five C2H2 zinc fingers. a-Helices are shown as cylinders, Zn+2 ions as spheres. Finger 1 does not interact with DNA, whereas the other four fingers do. (b) The glucocorticoid receptor is a homodimeric C4 zinc-finger protein. a-Helices are shown as purple ribbons, p-strands as green arrows, Zn+2 ions as spheres. Two a helices (darker shade), one in each monomer, interact with the DNA. Like all C4 zinc-finger homodimers, this transcription factor has twofold rotational symmetry; the center of symmetry is shown by the yellow ellipse. In contrast, heterodimeric nuclear receptors do not exhibit rotational symmetry. [See N. P Pavletich and C. O. Pabo, 1993, Science 261:1701, and B. F. Luisi et al., 1991, Nature 352:497.]

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