DNA-binding domains

Two domains, t1 and t2, exist which affect the GR post-DNA binding transcription activity (37). The major (t1) transactivation domain is 185 amino acid residues ia length with a 58-tesidue a-heUcal functional cote (38). The t1 domain is located at the N terminus of the proteia the minor (t2) trans activation domain residues on the carboxy-terminal side of the DNA binding domain. 

The x-ray structure of the DNA-binding domain of the lambda repressor is known... 

The x-ray structure of the N-terminal DNA-binding domain of the lambda repressor was determined to 3.2 A resolution in 1982 by Carl Pabo at Harvard University and revealed a structure with striking similarities to that of Cro, although the p strands in Cro are replaced by a helices in repressor. 

In spite of the absence of the C-terminal domains, the DNA-binding domains of lambda repressor form dimers in the crystals, as a result of interactions between the C-terminal helix number 5 of the two subunits that are somewhat analogous to the interactions of the C-terminal p strand 3 in the Cro protein (Figure 8.7). The two helices pack against each other in the normal way with an inclination of 20° between the helical axes. The structure of the C-terminal domain, which is responsible for the main subunit interactions in the intact repressor, remains unknown. 

This model of Cro binding to DNA was arrived at by intuition and clever model building. Its validity was considerably strengthened when the same features were subsequently found in the DNA-binding domains of the lambda-repressor molecule. The helix-turn-helix motif with a recognition helix is present in the repressor, and moreover the repressor DNA-binding domains dimerize in the crystals in such a way that the recognition helices are separated by 34 A as in Cro. 

By comparing the crystal structures of these complexes with a further complex of the 434 repressor DNA-binding domain and a synthetic DNA containing the operator region OR3, Harrison has been able to resolve at least in part the structural basis for the differential binding affinity of 434 Cro and repressor to the different 434 operator regions. 

Figure 8.11 The DNA-binding domain of 434 repressor. It is a dimer in its complexes with DNA fragments. Each subunit (green and brown) folds into a bundle of four a helices (1-4) that have a structure similar to the corresponding region of the lambda repressor (see Figure 8.7) including the helix-turn-helix motif (blue and red). A fifth a helix (5) is involved in the subunit interactions, details of which are different from those of the lambda repressor fragment. The structure of the 434 Cro dimer is very similar to the 434 repressor shown here.

The structures of 434 Cro and the 434 repressor DNA-binding domain are very similar... 

The 434 Cro molecule contains 71 amino acid residues that show 48% sequence identity to the 69 residues that form the N-terminal DNA-binding domain of 434 repressor. It is not surprising, therefore, that their three-dimensional structures are very similar (Figure 8.11). The main difference lies in two extra amino acids at the N-terminus of the Cro molecule. These are not involved in the function of Cro. By choosing the 434 Cro and repressor molecules for his studies, Harrison eliminated the possibility that any gross structural difference of these two molecules can account for their different DNA-binding properties. 

The tetrameric structure of the lac repressor has a quite unusual V-shape (Figure 8.22). Each arm of the V-shaped molecule is a tight dimer, which is very similar in structure to the PurR dimer and which has the two N-termi-nal DNA binding domains close together at the tip of the arm. The two dimers of the lac repressor are held together at the other end by the four carboxy-terminal a helices, which form a four-helix bundle. 

Steitz, T.A., et al. Structural similarity in the DNA-binding domains of catabolite gene activator and Cro repressor proteins. Proc. Natl. Acad. Sci. USA 79 3097-3100,... 

The two homologous repeats, each of 88 amino acids, at both ends of the TBP DNA-binding domain form two stmcturally very similar motifs. The two motifs each comprise an antiparallel p sheet of five strands and two helices (Figure 9.4). These two motifs are joined together by a short loop to make a 10-stranded p sheet which forms a saddle-shaped molecule. The loops that connect p strands 2 and 3 of each motif can be visualized as the stirmps of this molecular saddle. The underside of the saddle forms a concave surface built up by the central eight strands of the p sheet (see Figure 9.4a). Side chains from this side of the P sheet, as well as residues from the stirrups, form the DNA-binding site. No a helices are involved in the interaction area, in contrast to the situation in most other eucaryotic transcription factors (see below). 

The polypeptide chain of p53 is divided in three domains, each with its own function (Figure 9.16). Like many other transcription factors, p53 has an N-terminal activation domain followed by a DNA-binding domain, while the C-terminal 100 residues form an oligomerization domain involved in the formation of the p53 tetramers. Mutants lacking the C-terminal domain do not form tetramers, but the monomeric mutant molecules retain their sequence-specific DNA-binding properties in vitro. 

Figure 9.18 Schematic digram of the structure of the DNA-binding domain of p53. (a) The DNA binding domain of p53 folds into an antiparallel p barrel with long loop regions—...

Figure 9.19 Nucleotide sequence of the 21-base pair DNA fragment cocrystalUzed with the DNA-binding domain of p53. The p53 binds in a sequence-specific manner to the shaded region.

Decker, N., et al. Solution structure of the POU-specific DNA-binding domain of Oct-1. Nature 362 852-855, 1993. 

Two zinc-containing motifs in the glucocorticoid receptor form one DNA-binding domain... 

The two zinc ions fulfill important but different functions in the DNA-binding domains. The first zinc ion is important for DNA-bindlng because it properly positions the recognition helix the last two cysteine zinc ligands are part of this helix. The second zinc ion is important for dimerization since the five-residue loop between the first two cysteine zinc ligands is the main component of the dimer interaction area. 

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