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. 2013 Mar 1;27(5):552–564. doi: 10.1101/gad.210112.112

Figure 5.

Figure 5.

RapZ interacts with RNase E, which is essential for processing of GlmZ, and this interaction is independent of the RNA-binding function of RapZ. (A) Inactivation of RNase E abrogates processing of GlmZ in vivo. Northern blot analysis of total RNA isolated from strains N3433 (wild-type), N3431 (rneTS), IBPC633 (rnc-), and IBPC935 (rng-). Strain N3431 carries a temperature-sensitive RNase E variant, which becomes inactivated upon a temperature shift from 30°C to 44°C. Northern blot analysis was performed using the indicated probes. The loading controls are provided in Supplemental Figure S13. (B) RNase E copurifies with RapZ independently of RNA binding by RapZ. Strain Z64, which carries a Flag-tagged rne gene (encoding RNase E-Flag) on the chromosome and additionally contained either plasmid pBGG237 (empty vector; lanes 1–3), plasmid pBGG217 encoding Strep-PtsN (lanes 4–6), plasmid pBGG164 encoding Strep-RapZ (lanes 7–9), or plasmid pYG29 encoding Strep-RapZquad (lanes 10–13), was grown in LB containing 1 mM IPTG for induction of synthesis of the Strep-tagged proteins. The cell extracts were subjected to the copurification protocol using StrepTactin affinity chromatography (Supplemental Fig S11). The presence of RNase E-Flag in the eluates was tested by Western blotting using anti-Flag antiserum. (C) BACTH assays indicating interaction of the N-terminal part of RNase E with RapZ independent of its RNA-binding function. High β-galactosidase activities, which were monitored either quantitatively (left) or phenotypically on X-Gal agarose plates (right), reflect reconstitution of split adenylate cyclase CyaA activity though interaction of the proteins that are fused to its separately encoded T25 and T18 domains. The various plasmids that were tested in strain BTH101 are listed in Supplemental Table S2. (D) Dot blot far-Western indicating interaction of RapZ with the catalytic domain of RNase E in vitro. Various amounts of purified Strep-RapZ, Strep-RapZquad, and BSA (negative control) were spotted onto a membrane and subsequently incubated in 50 nM His6-tagged catalytic domain of RNase E. Interaction was visualized with an antiserum directed against the His tag.