Figure 6.

RapZ recruits GlmZ to processing by RNase E, and GlmY counteracts this reaction through sequestration of RapZ. (A) RapZ promotes cleavage of GlmZ by RNase E in vitro, and GlmY inhibits this process. In vitro cleavage assay of α-³²P-UTP-labeled GlmZ using varying concentrations of the catalytic domain of RNase E in the absence (lanes 1–4) or presence (lanes 5–12) of 150 nM Strep-RapZ. In lanes 9–12, unlabeled GlmY (60 nM) was additionally present. The asterisk indicates a nonspecific cleavage product of GlmZ. (B) RapZ triggers specific processing of GlmZ. In vitro cleavage assay of α-³²P-UTP-labeled GlmZ using 10 nM catalytic domain of RNase E and varying concentrations of Strep-RapZ. (C) Mutations in the lateral bulge inhibit processing of GlmZ by RNase E in vitro. In vitro cleavage assay of α-³²P-UTP-labeled GlmZ variants carrying the indicated mutations. Varying concentrations of the catalytic domain of RNase E and 150 nM Strep-RapZ were added as indicated. (D) GlmY and GlmZ compete for binding to RapZ in vivo. The effects of plasmid-driven overexpression of GlmY and GlmZ on sRNA copurification with Strep-RapZ were addressed. Strain Z479, which carried the arabinose-inducible strep-rapZ gene on the chromosome and either plasmid pYG23 for overexpression of glmY or plasmid pYG24 for overexpression of glmZ was grown in LB containing arabinose. (Bottom panel) The cell extracts were subjected to the copurification protocol using StrepTactin affinity chromatography, resulting in purification of Strep-RapZ (Western blot). (Top and middle panels) Coeluting RNA was subjected to Northern analysis. Five micrograms of total RNA of strain Z479 served as positive control (first lanes, respectively). The dotted line indicates cropping of lanes from the original blot. (E) Intracellular GlcN6P depletion in a glmS mutant shifts the proportion of the two sRNA that are bound to Strep-RapZ toward GlmY. Strains Z479 (lanes 4,5) and the isogenic ΔglmS mutant Z555 (lanes 6,7) lack the authentic rapZ gene but carry the arabinose-inducible strep-rapZ gene at an ectopic site. The strains were grown in LB containing GlcN and arabinose until an OD₆₀₀ = 0.3. Subsequently, the cultures were split, and growth was continued in the absence or presence of GlcN (Supplemental Fig. S21B). After 2 h, the cultures were subjected to the copurification protocol, resulting in purification of Strep-RapZ (Supplemental Fig. S23). The copurifying sRNAs were analyzed by Northern blotting. (Lanes 2,3) Wild-type strain R1279 carrying no strep-rapZ served as negative control. (Lane 1) Five micrograms of total RNA of strain R1279 served as positive control.