Visualization of trp repressor and its complexes with DNA by atomic force microscopy

We used tapping mode atomic force microscopy to visualize the protein/prote in and the protein/DNA complexes involved in transcriptional regulation by the frp repressor (TR). Plasmid fragments bearing the natural operators Srp EDCBA and trp R, as well as nonspecific fragments, were deposited onto mic a in the presence of varying concentrations of TR and imaged. In the presen ce of L-tryptophan, both specific and nonspecific complexes of TR with DNA are apparent, as well as free TR assemblies directly deposited onto the mic a surface. We observed the expected decrease in specificity of TR for its o perators with increasing protein concentration (1-5 nM). This loss of DNA-b inding specificity is accompanied by the formation of large protein assembl ies of varying sizes on the mica surface, consistent with the known tendenc y of the repressor to oligomerize in solution. When the cc-repressor is omi tted, no repressor molecules are seen, either on the plasmid fragments or f ree on the mica surface, probably because of the formation of larger aggreg ates that are removed from the surface upon washing. All these findings sup port a role for protein/protein interactions as an additional mechanism of transcriptional regulation by the trp repressor.