The indirect read-out of DNA sequence by the P22 repressor protein
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The P22 repressor (P22R) is a transcriptional regulator produced by the temperate bacteriophage P22. In the bacteriophage, this repressor protein aids in establishing and maintaining the lysogenic state by simultaneously inhibiting the transcription of lytic genes and activating the transcription of the genes necessary for lysogeny. These functions require that P22R binds to six, eighteen base pair operator sites that are contained within two operator regions, O R and O L , on the phage chromosome. When P22R binds, it makes base specific contacts with certain bases in the outer edges of the binding sites however, it does not bind the four central bases of the operator. An analysis of the sequence composition of the six naturally occurring P22R sites reveal that the outermost bases that are contacted by repressor exhibit sequence conservation, while the sequence of the central bases are divergent. These central bases are not contacted by P22R however, their sequence affects P22R’s affinity through a mechanism called "indirect read-out" that is defined as the sensing of base pair identity without direct base-protein contact. Thus the indirect read-out of DNA sequence by P22R has a significant role in operator site discrimination by repressor and therefore its function as a regulator of transcription. The goal of this thesis is to elucidate the mechanism of indirect read-out of central sequence by P22R. When P22R binds its site, it induces transition from a B-DNA to a B’-DNA configuration in the non-contacted region of an operator site. B’-DNA is a unique and rare structural conformation that is characterized by a narrow minor groove and a spine of hydration. This spine of hydration contains a series of solvent molecules that are organized in layers and interconnected through hydrogen bonds. The work described herein was designed to test the hypothesis that the basis of the mechanism of indirect read-out by P22R is repressor’s induction of a B’-DNA conformation in the non-contacted bases of its operator sites. Consistent with this hypothesis, binding assays revealed that P22R’s affinity for operator sites correlates with ease by which this B’ structure can be imposed into the central region of the binding site. P22R apparently senses the structure of DNA at the center of its binding sites via negatively charges amino acid residues that are located close to but do not contact the central bases. My data suggests that these residues are "guardrails" that keep the minor groove of the non-contacted bases narrow and enable repressor to gauge the ease of B’ induction. This work has also revealed that the organization and coordination of the water molecules of the spine of hydration is profoundly important not only for the stability of the induced B’-DNA structure at the center of the binding site but also the entire P22R-operator complex.