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 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.
Joachimiak
A
A
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
Haran
T E
TE
Sigler
P B
PB
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA; D004926:Escherichia coli / Q000378:metabolism; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009875:Operator Regions, Genetic; D012097:Repressor Proteins / Q000378:metabolism*; D014364:Tryptophan / Q000096:biosynthesis*; D014867:Water / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D004247:DNA; D012097:Repressor Proteins; D014364:Tryptophan; D014867:Water; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins