Visualization of trp repressor and its complexes with DNA by atomic force microscopy.
We used tapping mode atomic force microscopy to visualize the protein/protein and the protein/DNA complexes involved in transcriptional regulation by the trp repressor (TR). Plasmid fragments bearing the natural operators trp EDCBA and trp R, as well as nonspecific fragments, were deposited onto mica in the presence of varying concentrations of TR and imaged. In the presence of L-tryptophan, both specific and nonspecific complexes of TR with DNA are apparent, as well as free TR assemblies directly deposited onto the mica surface. We observed the expected decrease in specificity of TR for its operators with increasing protein concentration (1-5 nM). This loss of DNA-binding specificity is accompanied by the formation of large protein assemblies of varying sizes on the mica surface, consistent with the known tendency of the repressor to oligomerize in solution. When the co-repressor is omitted, no repressor molecules are seen, either on the plasmid fragments or free on the mica surface, probably because of the formation of larger aggregates that are removed from the surface upon washing. All these findings support a role for protein/protein interactions as an additional mechanism of transcriptional regulation by the trp repressor.
Margeat
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E
Centre de Biochimie Structurale, INSERM U 414-CNRS UMR 9955-Université Montpellier I, Montpellier, France.
Le Grimellec
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C
Royer
C A
CA
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D018625:Microscopy, Atomic Force / Q000379:methods*; D009875:Operator Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D046911:Macromolecular Substances; D012097:Repressor Proteins; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins