The RssB response regulator directly targets ςS for degradation by ClpXP

Abstract

The ς S subunit of Escherichia coli RNA polymerase regulates the expression of stationary phase and stress response genes. Control over ς S activity is exercised in part by regulated degradation of ς S. In vivo, degradation requires the ClpXP protease together with RssB, a protein homologous to response regulator proteins. Using purified components, we reconstructed the degradation of ς S in vitro and demonstrate a direct role for RssB in delivering ς S to ClpXP. RssB greatly stimulates ς S degradation by ClpXP. Acetyl phosphate, which phosphorylates RssB, is required. RssB participates in multiple rounds of ς S degradation, demonstrating its catalytic role. RssB promotes ς S degradation specifically; it does not affect degradation of other ClpXP substrates or other proteins not normally degraded by ClpXP. ς S and RssB form a stable complex in the presence of acetyl phosphate, and together they form a ternary complex with ClpX that is stabilized by ATP[γ-S]. Alone, neither ς S nor RssB binds ClpX with high affinity. When ClpP is present, a larger ς S–RssB–ClpXP complex forms. The complex degrades ς S and releases RssB from ClpXP in an ATP-dependent reaction. Our results illuminate an important mechanism for regulated protein turnover in which a unique targeting protein, whose own activity is regulated through specific signaling pathways, catalyzes the delivery of a specific substrate to a specific protease.