IN-VITRO PHOSPHORYLATION STUDY OF THE ARC 2-COMPONENT SIGNAL-TRANSDUCTION SYSTEM OF ESCHERICHIA-COLI

The ArcB and ArcA proteins constitute a two-component signal transduct ion system that plays a broad role in transcriptional regulation, Unde r anoxic or environmentally reducing conditions, the sensor kinase (Ar cB) is stimulated to autophosphorylate at the expense of ATP and subse quently transphosphorylates the response regulator (ArcA). ArcB is a c omplex, membrane-bound protein comprising at least three cytoplasmic d omains, an N-terminal transmitter domain with a conserved His292 resid ue (H1), a central receiver domain with a conserved Asp576 residue (D1 ), and a C-terminal alternative transmitted-domain,vith a conserved Hi s717 residue (H2), To study the phosphoryl transfer pathways of the Ar e system, we prepared the following His-tagged proteins: H1, D1, H2, H 1-D1, D1-H2, H1-D1-H2, and ArcA. Incubations of various combinations o f Are proteins with [gamma-P-32]ATP indicated that H1, but not D1 or H 2, catalyzes autophosphorylation; that H1-P transfers the phosphoryl g roup to D1 much more rapidly than to ArcA; and that D1 accelerates the transphosphorylation of H2. Finally, ArcA is phosphorylated much more rapidly by H2-P than by H1-P, Available data are consistent with a si gnal transduction model in which (i) reception of a membrane signal(s) triggers autophosphorylation of H1 at His292, (ii) the phosphoryl gro up can migrate to D1 at Asp576 and subsequently to H2 at His717, and ( iii) ArcA receives the phosphoryl group from either His292 or His717, the relative contribution of which is regulated by cytosolic effecters .