Repressor DNA-binding domain

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. 

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

Figure 31.6. LAC Repressor-DNA Interactions. The lac repressor DNA-binding domain inserts an a helix into the major groove of operator DNA. A specific contact between an arginine residue of the repressor and a G-C base pair is shown at the right.

D. W. Rodgers and S. C. Harrison, The complex between phage 434 repressor DNA-binding domain and operator site OR3 stmctural differences between consensus and non-consensus half-sites. Structure, 1 (1993), 227. 

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. 

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 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 three-dimensional strucmres of Cro and of the lambda repressor protein have been determined by x-ray crystallography, and models for their binding and effecting the above-described molecular and genetic events have been proposed and tested. Both bind to DNA using hehx-turn-helix DNA binding domain motifs (see below). 

Anderson, J. E., Ptashne, M. and Harrison, S. C. (1984). Cocrystals of the DNA-binding domain of phage 434 repressor and a synthetic phage 434 operator. Proc. Natl. Acad. Sci. USA 81,1307-1308. 

Direct repressors interact with the basal components of the transcription apparatus or with transcriptional activators to inhibit their activity. Specific repressors, analogous to transcriptional activators, are constructed modularly, with a DNA-binding domain and a repressor domain. The repressive character of such domains has been proven in domain swapping experiments. The mechanism of specific repression remains speculative. The following mechanisms are, however, conceivable ... 

Figure 28-3 (A) Ribbon view of the dimeric lac repressor bound to a natural operator and to the anti-inducer o-nitro-phenylfucoside (ONPF). The headpiece (residues 2-46) and the hinge helix (residues 50-58) form the DNA-binding domains. The core (residues 62-330), which is divided into N- and C-terminal subdomains, forms the binding site for ONPF. The C-terminal residues 334-360, which form a tetramerization domain, are absent from this MolScript drawing. Notice that the hinge helices bind to and widen the minor groove at the center of the operator. From Lewis et al.5a (B) Model of a 93-bp DNA loop corresponding to residues -82 to +11 of the lac operon (Fig. 28-2) bound to the tetrameric lac repressor. The active sites of the repressor are bound to the major operator O, and to the secondary operator 03. From Lewis et al.5...

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