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. 2001 Mar 20;98(7):3768–3772. doi: 10.1073/pnas.071043698

Figure 2.

Figure 2

Fluorescently labeled lambda repressor bearing the COOH-terminal 11-residue ssrA tag (Fl-λRssrA) forms a stable complex with ClpA hexamers in ATPγS and associates stably with mature, inactive ClpP (miClpP) following ATP-induced translocation. (a) Gel-filtration profiles of Fl-λR21CssrA and ClpA in the presence and absence of 0.5 mM ATPγS, monitored by absorbance at 229 nm (Upper) and by fluorescein fluorescence (Lower). ClpA (3 μM hexamer) was incubated with 2 μM Fl-λR21CssrA in Clp reaction buffer plus 1 mM ATPγS for 1 h. Reactions were analyzed by gel filtration on a Superose 12 column. (b) Gel-filtration profile of Fl-λR21CssrA encapsulated in miClpP after ATP-driven translocation from ClpA, monitored by fluorescein fluorescence. ClpA (2 μM), Fl-λR21CssrA (2 μM), and miClpP (8 μM) were incubated for 15 min at room temperature in Clp reaction buffer plus 10 mM ATP before Superose 12 chromatography in reaction buffer containing 1 mM ATP. The elution position of miClpP/Fl-λRssrA was confirmed by SDS/PAGE analysis of column fractions (not shown). The majority of ClpA was found in the ternary complex with miClpP and Fl-λRssrA (ClpA/miClpP/Fl-λRssrA).