DIFFERENTIAL RECOGNITION OF O(R)1 AND O(R)3 BY BACTERIOPHAGE-434 REPRESSOR AND CRO

The developmental decisions of bacteriophage 434 depend on the ability of 434 repressor and Cro to bind O(R)1 and O(R)3 with different relat ive affinities; repressor binds O(R)1 tighter than O(R)3, whereas Cro slightly prefers O(R)3 over O(R)1. Studies with operator mutants show that repressor's lower relative affinity for O(R)3 results from a devi ation in the sequence of O(R)3 from consensus; an A --> G change at po sition 4 in one half-site (O(R)1: A-C-A-A-A-C-T-T-T-C-T-T-G-T; O(R)3: A-C-A-G-T-T-T-T-T-C-T-T-G-T). Similar experiments show that Cro binds operators containing either A.T or G.C bases pairs at position 4 equal ly well, but cannot bind operators containing C.G or T.A base pairs at this position. A Gln33 --> Ala mutation in 434 repressor diminishes, but does not eliminate, its ability to distinguish between purines at position 4. This shows that a glutamine at amino acid 33 is not the so le determinant of repressor's position 4 specificity. Changing Gln33 - -> Leu. the amino acid at the homologous position in Cro, does not con fer ''Cro-like'' position 4 base specificity on repressor. Similarly, a Cro protein bearing Gln at this position does not exhibit repressor' s position 4 base preferences. The residual specificities of these mut ant proteins indicates that in each protein, more than 1 amino acid is responsible for recognizing bases at position 4. These were identifie d by analyzing the binding specificities of multiply mutated repressor s, in vitro. The types of substitutions made were guided by sequence h omologies between 434 repressor and Cro. At least three mutations are needed to eliminate repressor's position 4 base specificity; Gln33 --> Ala, Glu32 --> Gln, and Thr27 --> Lys, although no set of amino acid substitutions in repressor was able to confer Cro-like position 4 spec ificity to repressor. These results indicate that at least the amino a cids at these positions are involved in recognition of the position 4 base. Other evidence suggests that Cro and repressor use identical ami no acids present at homologous positions in the DNA recognition helix in different ways.