Role of protein--protein interactions in the regulation of transcription by trp repressor investigated by fluorescence spectroscopy.
In the present work, we have characterized the protein--protein interactions in the trp repressor (TR) from Escherichia coli using fluorescence spectroscopy. The steady-state and time-resolved fluorescence anisotropy of repressor labeled with 5-(dimethylamino)naphthalene-1-sulfonamide (DNS) was used to monitor subunit equilibria in the absence and presence of corepressor. In the absence of tryptophan, the repressor is in equilibrium between tetramers and dimers in the concentration range studied (approximately 0.04-40 microM in dimer). Binding of corepressor resulted in a marked destabilization of the tetramer. The beginning of a dimer-monomer dissociation transition was observed by monitoring the decrease in the intrinsic tryptophan emission energy upon dilution below 0.1 microM in dimer, indicating an upper limit for the dimer-dissociation constant near 1 nM. DNA titrations with a 26 base pair sequence containing the trp EDCBA operator performed in the absence and presence of the corepressor are consistent with a 1:1 dimer/operator stoichiometry in the presence of tryptophan, while the aporepressor binds with TR dimer/DNA stoichiometries greater than one and which depend upon both the concentration of protein and that of the operator. Using the multiple observable parameters available in fluorescence, we have thus carried out a thorough investigation of the coupled equilibria in this bacterial repressor. Our results are consistent with a physiologically relevant thermodynamic role for tetramerization in the regulatory function of the trp repressor. The present results which have brought to light novel protein--protein interactions in the trp repressor system indicate that fluorescence spectroscopic methods could prove quite useful in the study of the role of protein--protein interactions in eukaryotic systems as well.
Fernando
T
T
School of Pharmacy, University of Wisconsin-Madison 53706.
Royer
C
C
D001483:Base Sequence; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003619:Dansyl Compounds; D004926:Escherichia coli / Q000235:genetics*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D015964:Gene Expression Regulation, Bacterial; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009876:Operon; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D014158:Transcription, Genetic; D014364:Tryptophan / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D012097:Repressor Proteins; C001026:5-dimethylaminonaphthalene-1-sulfonamide; D014364:Tryptophan