A LacI-DNA Looping Landscape and Allosteric Effects on the Loop Shapes

Abstract

The lac operon and its repressor (LacI) are the prototype model for gene regulation. LacI negatively regulates lacZYA by binding a primary DNA operator site overlapping the promoter, and repression is enhanced by secondary operators that deliver LacI via looping intervening DNA. LacI's ability to form stable loops with a variety of DNA lengths has been attributed to protein flexibility and/or to multiple loop topologies. Previously developed DNA constructs in which looping is hyperstabilized by an A-tract bend placed between two operators provide different loop shapes depending on the operator/bend helical phasing. Here, FRET is used to characterize the sequence/structure landscape of a set of related constructs with systematically varied operator/bend phasings. Donor and acceptor fluorophores positioned on either side of the operator provide multiple distance constraints on the orientations of the LacI-DNA loop. The results suggest that LacI can form many different looped states whose relative energetics can be measured. Also, IPTG addition demonstrates that inducer-bound LacI still forms stable loops, probably with different geometries relative to the repressed state. This comprehensive looping landscape should allow determination of whether protein flexibility is necessary to explain the results.