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

Salmonella bacteriophage repressor Mnt belongs to the ribbon-helix-helix cl ass of transcription factors. Previous SELEX results suggested that interac tions of Mnt with positions 16 and 17 of the operator DNA are not independe nt. Using a newly developed high-throughput quantitative multiple fluoresce nce relative affinity (QuMFRA) assay, we directly quantified the relative e quilibrium binding constants (K-ref) of Mnt to operators carrying all the p ossible 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 mu tants, demonstrating the non-independence of these two positions. The abili ty to produce a large number of quantitative binding data simultaneously an d the potential to scale up makes QuMFRA a valuable tool for the large-scal e study of macromolecular interaction.