HOW 434-REPRESSOR DISCRIMINATES BETWEEN O(R)1 AND O(R)3 - THE INFLUENCE OF CONTACTED AND NONCONTACTED BASE-PAIRS

The sequence of the bacteriophage 434 O(R)1 (ACAAAACTTTCTTGT) differs from its O(R)3 (ACAGTTTTTCTTGT) at positions 4-6. X-ray analysis shows that the side chain of Gln(33) of the 434 repressor makes van der Waa ls' and H-bond contacts with the T at position 4' in complex with O(R) 1, but no specific contact is observed at this position in 434 repress or-O(R)3 complexes, No contacts are made by repressor to the bases at positions 5 or 6 in either binding site, The significance of the seque nce differences between O(R)1 and O(R)3 in determining the operator af finity for repressor were examined by con structing synthetic variants of these operators, Measurements of the affinity of these operators f or repressor as a function of ionic strength revealed that although ba se pairs 5 and 6 are not contacted by 434 repressor, they can nonethel ess influence operator affinity for repressor by modulating the degree to which ionic interactions contribute to the overall binding energy. Both the magnitude and direction of their effect depends on the statu s of repressor's contacts to the bases at position 4. The role of cont act made by Gln(33) to position 4 was examined by mutating this amino acid to Ala and by examining the affinity of wild type repressor for a n operator bearing a 5-methylcytosine at position 4' in an O(R)1-4G mu tant. These experiments showed that repressor's preferences at operato r positions 5 and 6 are linked to its position 4 preference via a van der Waals' contact between amino acid 33 and a methyl group on the bas e at operator position 4'. Together, the results of the experiments sh own here reveal that bases that do not contact the protein alter its p references for bases at the contacted operator position 4.