Non-independence of Mnt repressor-operator interaction determined by a new quantitative multiple fluorescence relative affinity (QuMFRA) assay

Salmonella bacteriophage repressor Mnt belongs to the ribbon-helix-helix class of transcription factors. Previous SELEX results suggested that interactions of Mnt with positions 16 and 17 of the operator DNA are not independent. Using a newly developed high-throughput quantitative multiple fluorescence relative affinity (QuMFRA) assay, we directly quantified the relative equilibrium binding constants (K-ref) of Mnt to operators carrying all the possible dinucleotide combinations at these two positions. Results show that Mnt prefers binding to C, instead of wildtype A, at position 16 when wild-type C at position 17 is changed to other bases. The measured K-ref values of double mutants were also higher than the values predicted from single mutants, demonstrating the non-independence of these two positions. The ability to produce a large number of quantitative binding data simultaneously and the potential to scale up makes QuMFRA a valuable tool for the large-scale study of macromolecular interaction.