The repressor of phage 434 binds to six operator sites on the phage chromosome. A comparison of the sequences of these 14-base-pair (bp) operator sites reveals a striking pattern: at five of the six sites, the symmetrically arrayed outer eight base pairs (four in each half-site) are identical and the remaining site differs at only one position (Fig. 1b). In contrast, the sequences of the inner four base pairs are highly variable. Crystallographic analysis of the repressor-operator complex shows that at each half-site, the 'recognition alpha-helix' of the repressor is positioned in the major groove such that it could contact the outermost five base pairs, but not the innermost two (Fig. 1a). We show in this paper that the sequence of the central base pairs of the operator (two in each half-site) have a significant role in determining operator affinity for repressor, despite the evidence presented here and in the accompanying paper that these base pairs are not contacted by repressor. We also show that these central base pairs influence operator affinity for Cro, a second gene regulatory protein encoded by phage 434. We discuss the likely structural basis for this evidently indirect, but sequence-dependent, effect of the central base pairs of the operator on its affinity for the two regulatory proteins.

Bacterially derived exotoxins kill eukaryotic cells by inactivating factors and/or pathways that are universally conserved among eukaryotic organisms. The genes that encode these exotoxins are commonly found in bacterial viruses (bacteriophages). In the context of mammals, these toxins cause diseases ranging from cholera to diphtheria to enterohemorrhagic diarrhea. Phage-carried exotoxin genes are widespread in the environment and are found with unexpectedly high frequency in regions lacking the presumed mammalian "targets," suggesting that mammals are not the primary targets of these exotoxins. We suggest that such exotoxins may have evolved for the purpose of bacterial antipredator defense. We show here that Tetrahymena thermophila, a bacterivorous predator, is killed when cocultured with bacteria bearing a Shiga toxin (Stx)-encoding temperate bacteriophage. In cocultures with Tetrahymena, the Stx-encoding bacteria display a growth advantage over those that do not produce Stx. Tetrahymena is also killed by purified Stx. Disruption of the gene encoding the StxB subunit or addition of an excess of the nontoxic StxB subunit substantially reduced Stx holotoxin toxicity, suggesting that this subunit mediates intake and/or trafficking of Stx by Tetrahymena. Bacterially mediated Tetrahymena killing was blocked by mutations that prevented the bacterial SOS response (recA mutations) or by enzymes that breakdown H 2 O 2 (catalase), suggesting that the production of H 2 O 2 by Tetrahymena signals its presence to the bacteria, leading to bacteriophage induction and production of Stx.

The P22 c2 repressor protein (P22R) binds to DNA sequence-specifically and helps to direct the temperate lambdoid bacteriophage P22 to the lysogenic developmental pathway. We describe the 1.6 A X-ray structure of the N-terminal domain (NTD) of P22R in a complex with a DNA fragment containing the synthetic operator sequence [d(ATTTAAGATATCTTAAAT)]2. This operator has an A-T base pair at position 9L and a T-A base pair at position 9R and is termed DNA9T. Direct readout: nondirectional van der Waals interactions between protein and DNA appear to confer sequence-specificity. The structure of the P22R NTD-DNA9T complex suggests that sequence-specificity arises substantially from lock-and-key interaction of a valine with a complementary binding cleft on the major groove surface of DNA9T. The cleft is formed by four methyl groups on sequential base pairs of 5'-TTAA-3'. The valine cleft is intrinsic to the DNA sequence and does not arise from protein-induced DNA conformational changes. Protein-DNA hydrogen bonding plays a secondary role in specificity. Indirect readout: it is known that the noncontacted bases in the center of the complex are important determinants of affinity. The protein induces a transition of the noncontacted region from B-DNA to B'-DNA. The B' state is characterized by a narrow minor groove and a zigzag spine of hydration. The free energy of the transition from B- to B'-DNA is known to depend on the sequence. Thus, the observed DNA conformation and hydration allows for the formulation of a predictive model of the indirect readout phenomenon.

The affinity of the Eseherichia coli phage 434 operator for phage 434 repressor is affected by changes in the sequence of the noncontacted base pairs near the operator's center. The results presented here show that base composition near the center ofthe operator affects the operator's affinity for repressor by altering the ease with which the operator can be overtwisted into the proper configuration for complex formation. We show that both DNA flexibility and repressor flexibility influence the strength of the repressor-operator interaction: an operator with a single-strand nick at its center has a higher affinity for repressor than does the intact operator: and a repressor bearing a mutation that results in a relaxed dimer interaction is less sensitive than is wild type to changes in the flexibility of the operator. We show that the effect of noncontacted base pairs on operator affinity is independent of the slight overall bend of the operator seen in the repressoroperator complex. Central sequence effects on affinity for repressor are independent ofthe identity ofadjacent base pairs, suggesting that the structure of the individual base pairs, not interactions between them, are responsible for the different torsional rigidities of different operators.Our current picture of how the Escherichia coli phage 434 repressor recognizes its DNA operator is summarized in Fig. 1. In the repressor-operator complex, one a-helix of the repressor dimer lies in each half-site of the operator. Each of these helices is positioned in the major groove so that its side chains can contact the outermost 5 base pairs in one operator half-site (1). Biochemical and x-ray crystallographic studies suggest that neither this "recognition helix" nor any other part of the repressor contacts the innermost 2 base pairs of each half-site (1, 2). Nevertheless, the operator's affinity for repressor is determined, in part, by the composition of these base pairs: operators bearing APT or T'A base pairs at operator positions 6-9 bind the repressor more strongly than do operators bearing G-C or C-G base pairs at these positions (2). We have proposed that the base composition near the center of the operator affects its affinity for repressor by altering the ease with which operator DNA can be deformed into the optimal configuration for complex formation (2). Crystallographic analysis of the 434 repressor-operator complex shows that when 434 operator binds to 434 repressor, the DNA near the center of the operator is slightly overtwisted, so that the minor groove in this region is compressed (Fig. 1) (1). Evidently this DNA deformation is required to align the two operator half-sites so that each monomer of the bound repressor dimer can make optimal contacts with each half-site.In this paper we explore more directly the role of both DNA flexibility and repressor flexibility in determining the affinity of operator for repressor. We reasoned that introducing a single-strand break at the central phosphodiester bond of the operator would increase th...