Search

A method to rapidly screen libraries of cyclic peptides in vivo for molecules with biological activity has been developed and used to isolate cyclic peptide inhibitors of the ClpXP protease. Fluorescence activated cell sorting was used in conjunction with a fluorescent reporter to isolate cyclic peptides that inhibit the proteolysis of tmRNA-tagged proteins in Escherichia coli. Inhibitors shared little sequence similarity and interfered with unexpected steps in the ClpXP mechanism in vitro. One cyclic peptide, IXP1, inhibited the degradation of unrelated ClpXP substrates and has bactericidal activity when added to growing cultures of Caulobacter crescentus, a model organism that requires ClpXP activity for viability. The screen used here could be adapted to identify cyclic peptide inhibitors of any enzyme that can be expressed in E. coli in conjunction with a fluorescent reporter.

The production and removal of regulatory RNAs must be controlled to ensure proper physiological responses. SsrA RNA (tmRNA), a regulatory RNA conserved in all bacteria, is cell cycle regulated and is important for control of cell cycle progression in Caulobacter crescentus. We report that RNase R, a highly conserved 3' to 5' exoribonuclease, is required for the selective degradation of SsrA RNA in stalked cells. Purified RNase R degrades SsrA RNA in vitro, and is kinetically competent to account for all SsrA RNA turnover. SmpB, a tmRNA-binding protein, protects SsrA RNA from RNase R degradation in vitro, and the levels of SmpB protein during the cell cycle correlate with SsrA RNA stability. These results suggest that SmpB binding controls the timing of SsrA RNA degradation by RNase R. We propose a model for the regulated degradation of SsrA RNA in which RNase R degrades SsrA RNA from a non-tRNA-like 3' end, and SmpB specifically protects SsrA RNA from RNase R. This model explains the regulation of SsrA RNA in other bacteria, and suggests that a highly conserved regulatory mechanism controls SsrA activity.