AFM Studies of λ Repressor Oligomers Securing DNA Loops

Large, cooperative assemblies of proteins that wrap and/or loop genomic DNA may epigenetically shift configurational equilibria that determine developmental pathways. Such is the case of the lambda bacteriophage which may exhibit virulent (lytic) or quiescent (lysogenic) growth. The lysogenic state of lambda prophages is maintained by the lambda repressor (CI), which binds to tripartite operator sites in each of the O-L and O-R control regions located about 2.3 kbp apart on the phage genome and represses lytic promoters. Dodd and collaborators have suggested that an initial loop formed by interaction between CI bound at O-R and O-L provides the proper scaffold for additional CI binding to attenuate the P-RM promoter and avoid over production of CI. Recently, the looping equilibrium as a function of CI concentration was measured using tethered particle motion analysis, but the oligomerization of CI in looped states could not be determined. Scanning force microscopy has now been used to probe these details directly. An equilibrium distribution of looped and unlooped molecules confined to a plane was found to be commensurate to that for tethered molecules in solution, and the occupancies of specific operator sites for several looped and unlooped conformations were determined. Some loops appeared to be sealed by oligomers of 6-8, most by oligomers of 10-12, and a few by oligomers of 14-16.