MUTAGENESIS SUPPORTS WATER MEDIATED RECOGNITION IN THE TRP REPRESSOR-OPERATOR SYSTEM

High resolution crystallographic analysis of the trp repressor - operator complex indicates that the principal determinants of specificity are water mediated hydrogen bonds between the helix-turn-helix and the identity elements of the operator. One such hydration site involves a conserved G-C base pair (designated G(6)) Six nucleotides away from the dyad which, if changed symmetrically to any other pair (e.g. G(6) --> A) reduces affinity to nonspecific levels. This same water site also contacts the conserved A(5) which, if changed to G (mutation A(5) --> G), also diminishes affinity. The stereochemistry of the water mediated hydrogen bonding system predicts that the severe deterioration of in vitro binding caused by G(6) --> A Should be reverted by a second deleterious mutation A(5) --> G. This proved to be the case. No other second mutation at conserved operator position 5 or 7 (flanking the G(6) --> A) reversed the effect of G(6) --> A.