Mutations in the RNase H-like and DNase I domains improve catalytic efficiency of RNase E. (A) The left shows a schematic of secondary structure of 9S RNA with three cleavage sites marked as “a,” “b,” and “c” (Christiansen 1988; Lorenz et al. 2011); the bars above the schematic show the three segments (9S-V1, 9S-V2, and 9S-V3) generated for cleavage assays. The middle panel shows secondary structure of GlmZ RNA predicted by the ViennaRNA Package 2.0 (Lorenz et al. 2011). The right panel shows an annotated domain schematic for NTD-wt and NTD-3M harboring mutations in RNase H-like (D26N and D28N) and DNase I (D338N) domains. (B) Denaturing RNA gels showing time course cleavage assay of 9S (5′-triphosphorylated, upper panel; 5′-monophosphorylated, lower panel) using NTD-wt (blue lines) and the NTD-3M (red lines). The lower panel shows the integrated signal for 9S (left) and p5S product (right). (C) Integrated signal for the 9S segments V1, V2, and V3 obtained against NTD-wt and NTD-3M. (D) Integrated signal for GlmZ cleavage over time for NTD-wt and NTD-3M shown on the left panel with the corresponding denaturing gels shown on the right. (E) Denaturing RNA gels for GlmZ processing by NTD-wt and NTD-3M in the presence of RapZ or Hfq, showing the production of GlmZ-Pro is sensitive to the presence of RapZ but not Hfq. (F) Michaelis–Menten plots used to determine the kinetics parameters of cleavage of 9S and GlmZ RNAs. The plots were fitted using Prism (GraphPad Software) and represent mean of three representative plots of reaction rates versus substrate concentrations (see “Materials and Methods” for details). (H) RNase H-like domain, (S1) RNA binding S1 domain, (DNase I) DNase I-like domain, (5′) RNA 5′ site-sensing pocket, (Zn) Zn-linker.
