Ligand-induced self-association of the Escherichia coli regulatory protein TyrR.

Analyses of the sedimentation properties of the Escherichia coli regulatory protein TyrR indicated that it undergoes a ligand-induced hexamerization. This phenomenon was observed at protein concentrations approximating to those found in vivo. In the absence of added ligands, TyrR sedimented as a single molecular species with a sedimentation coefficient of 5.3 S and a relative molecular mass of 113,000. Given a subunit relative molecular mass of 57,640 for TyrR, it was concluded that this species is a dimer. Similar sedimentation properties were observed when TyrR was sedimented in the presence of either tyrosine, phenylalanine, ATP or ATP gamma S, a non-hydrolysable analogue of ATP. However, in the presence of saturating ATP gamma S and 500 microM tyrosine or 25 mM phenylalanine the sedimentation behaviour of TyrR yielded relative molecular masses of 340,000 and 310,000, respectively, indicative of hexamer formation. The sedimentation data obtained across a range of TyrR concentrations fitted equally well to dimer-hexamer and dimer-tetramer-hexamer models. For the latter model, the predicted overall association constant was 3.2 x 10(13) M-2 at saturating tyrosine, while the relative values of the association constants for the two individual steps indicated a concerted mechanism with the tetramer a minor component. There was no indication of dimer dissociation when dilute TyrR solutions (100 nM) were sedimented. A model to explain the role of hexamerization in tyrosine-mediated repression of transcription by TyrR is proposed. It is suggested that the hexameric form of TyrR is the active repressing species, interacting with two or three specific sequences (TyrR boxes) in the targeted regulatory DNA. The hexamerization reaction that takes place when the tyrosine concentration rises is envisaged as occurring in situ on the DNA, with a TyrR dimer that permanently occupies one of the TyrR boxes acting as a nucleation site for the development of the hexamer-DNA complex.