Mutagenesis supports water mediated recognition in the trp repressor-operator system. |
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| Authors: | A Joachimiak T E Haran and P B Sigler |
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| Affiliation: | Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511. |
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| Abstract: | 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 G6) six nucleotides away from the dyad which, if changed symmetrically to any other pair (e.g. G6-->A) reduces affinity to nonspecific levels. This same water site also contacts the conserved A5 which, if changed to G (mutation A5-->G), also diminishes affinity. The stereochemistry of the water mediated hydrogen bonding system predicts that the severe deterioration of in vitro binding caused by G6-->A should be reverted by a second deleterious mutation A5-->G. This proved to be the case. No other second mutation at conserved operator position 5 or 7 (flanking the G6-->A) reversed the effect of G6-->A. |
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