Differential recognition of OR1 and OR3 by bacteriophage 434 repressor and Cro

The developmental decisions of bacteriophage 434 depend on the ability of 434 repressor and Cro to bind OR1 and OR3 with different relative affinities; repressor binds OR1 tighter than OR3, whereas Cro slightly prefers OR3 over OR1. Studies with operator mutants show that repressor's lower relative affinity for OR3 results from a deviation in the sequence of OR3 from consensus; an A-->G change at position 4 in one half-site (OR1: A-C-A-A-A-C-T-T-T-C-T-T-G-T; OR3: 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 equally 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 sole determinant of repressor's position 4 specificity. Changing Gln33-->Leu, the amino acid at the homologous position in Cro, does not confer "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 mutant proteins indicates that in each protein, more than 1 amino acid is responsible for recognizing bases at position 4. These were identified by analyzing the binding specificities of multiply mutated repressors, in vitro. The types of substitutions made were guided by sequence homologies 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 specificity to repressor. These results indicate that at least the amino acids at these positions are involved in recognition of the position 4 base. Other evidence suggests that Cro and repressor use identical amino acids present at homologous positions in the DNA recognition helix in different ways.