Repressor protein binding sites

Figure 8.1 A region of DNA in the related bacteriophages lambda, 434, and P22 that controls the switch for synthesis of new phage particles. Two structural genes are involved in this switch one coding for a repressor protein and one coding for the Cro protein. Between these genes there is an operator region (OR) that contains three protein binding sites—ORl, OR2, and OR3.

The lac operon is ordinarily subject to repression and is activated by the presence of an inducer, now known to be allolactose, D-Galp-(il— 6-d-G1c. However, in experimental work artificial inducers such as isopropyl-[5-o-thiogaIactoside (IPTG) are most often used. Jacob and Monod postulated that the free repressor protein binds to the operator. In the presence of the inducer a conformational change takes place, destroying the affinity of the repressor protein for the operator site. Thus, in the presence of inducer the operator is not blocked, and the transcription takes place. Such an operon is said to be negatively controlled and inducible. 

The organism senses whether glucose is available by another regulatory mechanism which cooperates with the lac repressor and the lac operator. The promoter is therefore sub-divided into two specific regions, each of distinctive function. One is the RNA polymerase entry site, where RNA polymerase first becomes bound to DNA (cf. DNA transcription, Appendix 5.6), and the other is the protein binding site for the catabolite activator protein (CAP) (Fig. 5.39). The CAP protein binding site controls the polymerase site which, when bound to the DNA, allows successful transcription provided that the repressor is not bound. When the CAP protein is not bound, then RNA polymerase cannot bind and transcription cannot take place. 

Operator A DNA site where a repressor protein binds to block the initiation of transcription from an adjacent promoter. 

The fundamental unit of tertiary structure is the domain. A domain is defined as a polypeptide chain or a part of a polypeptide chain that can fold independently into a stable tertiary structure. Domains are also units of function. Often, the different domains of a protein are associated with different functions. For example, in the lambda repressor protein, discussed in Chapter 8, one domain at the N-terminus of the polypeptide chain binds DNA, while a second domain at the C-terminus contains a site necessary for the dimerization of two polypeptide chains to form the dimeric repressor molecule. 

The lysis-lysogeny decision depends upon which of the two promoters in the operator region is able to bind polymerase, and that, in turn, depends upon the binding of the Cro and repressor proteins to three binding sites—ORl, OR2, and OR3—in OR. These binding sites are situated in the middle of the operator in such a way that ORl and OR2 overlap the promoter... 

These genetic experiments clearly demonstrated that the proposed structural model for the binding of these proteins to the phage operators was essentially correct. The second a helix in the helix-turn-helix motif is involved in recognizing operator sites as well as in the differential selection of operators by P22 Cro and repressor proteins. However, a note of caution is needed many other early models of DNA-protein interactions proved to be misleading, if not wrong. Modeling techniques are more sophisticated today but are still not infallible and are certainly not replacements for experimental determinations of structure. 

The crystal structures of DtxR and IdeR provide a detailed picture of this protein family (Figure 3.7, Plate 5). The N-terminal domain (residues 1-73) containing a helix-turn-helix motif binds a recognition nucleotide sequence of about 21 base pairs, as is nicely shown in a cocrystal of DNA and DtxR (Pohl et al., 1999). The central domain (74-140) has a function in dimerization the role of the third carboxy-terminal domain (141-230) is uncertain. Although metal-binding sites have been defined in these crystal structures, the mechanism by which metal binding causes the structural changes between apo- and holo-repressor is not clear. 

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