Figure 7.

YmcA-YlbF-YaaT does not regulate Spo0E activity in vitro, but does stimulate the phosphorelay. (A) The phosphorelay was reconstituted in vitro as described in Experimental procedures. The relay was allowed to run for 60 minutes, and then 50 nM of purified Spo0E was added to the reaction in the presence or absence of 2.4 µM complex. Samples were taken at the indicated time points. Because the phosphorelay reactions are reversible, total protein-associated radioactivity was plotted versus time. (B) The phosphorelay proteins were pre-incubated with buffer or with 2.4 µM YmcA-YlbF-YaaT complex to allow for potential interactions. The relay was initiated by the addition of ³²P-γ-ATP, and samples were collected at the indicated times. For each experiment, the bands were quantified as described in experimental procedures, and total radioactivity associated with the four phosphorelay proteins is shown. In each panel, a representative graph selected from several replicates is shown.