Trp repressor mutations alter DNA complex stoichiometry.

Abstract

We have examined the interaction of a series of mutant trp repressors with various operator DNA sequences using gel retardation. Binding to 40 base pairs (bp) TrpEDCBA operator yielded patterns distinct from the wild-type protein for superrepressors EK13, EK18, and EK49, with a protein-DNA complex of higher stoichiometry (three dimers/operator) than observed for wild-type repressor (two dimers/operator). This higher stoichiometry complex may contribute to the enhanced binding affinity and higher protein-operator stability observed for the superrepressors. In contrast, DN46 displayed the same complexes characteristic of the wild-type protein, although the complex of a single dimer with operator was more prominent in the DN46 binding pattern than wild-type despite higher apparent affinity of this protein for TrpEDCBA operator than wild-type protein. The binding of AV77 was indistinguishable from the wild-type protein. Similar patterns to that found for TrpEDCBA were also observed for the 40-bp aroH operator and symmetrized derivatives of TrpEDCBA for these superrepressors. Binding of EK13, EK18, and EK49 superrepressors to half-site DNAs, composed of 20 bp of TrpEDCBA sequence coupled with 20 bp of lac operator sequence, yielded 2:1 complex as the primary product with no detectable 3:1 complex; thus, two half-sites appear to be required for generation of the 3:1 complex. Mutation in the tryptophan-binding site can also generate higher order complexes with TrpEDCBA DNA as demonstrated by the binding of VA58; the presence of 3:1 complex with this protein was also dependent on the presence of two half-sites. In addition to effects of sequence changes in the protein, the ligand employed can influence the binding pattern, as demonstrated for EK49 and VA58 using 5-methyl-tryptophan; the 3:1 complex is produced more prominently and at lower protein concentration for both mutants. It is apparent from these data that binding of the trp repressor to DNA is influenced by the operator sequence, the nature of the corepressor, as well as interactions (perhaps involving the N-terminal regions) that occur within and between the dimeric structure of this protein.