In vivoandin vitroStudies of TrpR-DNA Interactions

The binding of tryptophan repressor (TrpR) to its operators was examined quantitatively usingin vitroandin vivomethods. DNA sequence requirements for 1:1 and tandem 2 :1 (TrpR : DNA) binding in various sequence contexts were studied. The results indicate that the optimal half-site sequence for recognition by one helix-turn-helix motif of one TrpR dimer is_{3′ CNTGA 5′}^{5′ GNACT 3′}, consistent with contacts observed by X-ray diffraction analysis of cocrystalline 1:1 and 2 :1 complexes. Half-sites can be paired to form a palindrome either by direct abutment, forming the nucleation site for a tandem 2 :1 complex, or with an 8-base-pair spacer, forming a 1:1 target. Dimethylsulfate (DMS) methylation-protection footprintingin vitroof 1:1 and 2 :1 complexes formed sequentially on the two unequal half-site pairs of thetrpEDCBA operator fromSerratia marcescensindicated an obligate hierarchy of site occupancy, with one half-site pair serving as the nucleation site for tandem binding. DMS footprinting ofEscherichia colioperatorsin vivoshowed that, over a wide range of intracellular TrpR concentration, thetrpEDCBA operator is occupied by three repressor dimers,aroH is occupied by two dimers, and the 1:1 binding mode is used on thetrpR operator. The coexistence of these distinct occupancy states implies that changes in protein concentration affect only the fractional occupancy of each operator rather than the binding mode, which is determined by the number of half-site sequences present in the operator region. Cooperativity of tandem complex formation measured by gel retardation using a symmetrized synthetic operator containing identical, optimal sites spaced as in natural operators was found to be modest, implying a maximum coupling free energy of ～?2 kcal/mol. On other sequences the apparent degree of cooperativity, as well as the apparent affinity, varied with sequence and sequence context in a manner consistent with the structural models and which suggests compensation between affinity and cooperativity as a mechanism that allows tolerance of operator sequence variation.