RNA elements that were used as model targets of Csps. (A) Properties of CspA and CspD. MG1655 cells harboring the indicated plasmids were grown in LB-ampicillin medium. At an OD600 of 0.3, 0.2 mM IPTG was added to cultures to induce Csps. Incubation was continued for 30 min. The protein sample was subjected to western blotting using anti-FLAG monoclonal antibody and anti-GroEL polyclonal antibodies. The RNA sample was subjected to northern blotting using probes for baxL-bax mRNA, gdx mRNA, or tmRNA. Relative RNA levels were calculated, with the RNA sample of CspD set to 1 for baxL-bax mRNA and CspA set to 1 for gdx mRNA. The results are averages of three independent experiments, with error bars representing the standard deviations. Statistical significance was calculated using an unpaired two-tailed Student's t-test. (***) P < 0.001. (B) Evaluation of target specificity. The specificity score was calculated by a formula in which the level of gdx mRNA relative to that seen for CspA (relative efficiency for gdx) was subtracted from the level of baxL mRNA relative to that seen for CspD (relative efficiency for baxL). The quantitative data in A were used to calculate each efficiency. The results are averages of three independent experiments, with error bars representing the standard deviations. (C) In vivo binding of CspA-FLAG and CspD-FLAG to baxL-bax mRNA and gdx mRNA. Crude extract was prepared from MG1655 cells harboring the indicated plasmids and subjected to the pull-down assay using anti-FLAG M2 magnetic beads, as described in Materials and Methods. (D) The ratio of (Bound) relative to (Crude extract) is shown; in the calculation for baxL-bax mRNA, the sum of the full-length and truncated (*) was used. The results are averages of three independent experiments, with error bars representing the standard deviations.
