Two basic patches on the surface of HigB mediate recognition of 16S rRNA helices 18, 30, and 31. (A) HigB basic residues that interact with 16S rRNA cluster along two exposed surfaces (blue ovals) and are depicted as sticks. The mRNA path is shown as a dotted line as modeled from the 70S-HigB ΔH92 precleavage state (PDB code 4YPB) and is the location of the HigB active site. (B) Weblogo depiction of 1000 HigB homologs of the HigB regions (residues 1–30 and 68–73) that interact with 16S rRNA (Crooks et al. 2004). Residues are colored by the following scheme: Polar (G,S,T,Y,C; green), neutral (Q, N; magenta), basic (K, R, H; blue), acidic (D, E; red), and hydrophobic (A, V, L, I, P, W, F, M; black). Proteus vulgaris HigB residues are shown on the x-axis and residues that directly contact the 16S rRNA are depicted in white with black highlight. The y-axis indicates the bits with the height of the amino acid proportional to its frequency at that position. The total height of the stack is down-weighted if there is high variability while highly conserved positions are depicted as taller. (C) E. coli BW25113 growth assays show that overexpression of wild-type (WT) HigB halts cell growth (pink line) while uninduced HigB allows growth (black dash). HigB basic residues that directly interact with 16S rRNA were mutated to alanine and their effect on E. coli growth was monitored by optical density (OD) at 600 nm over 6 h. HigB patch one residues (K6, K8, and K11) were singly mutated and then doubly mutated in the sensitized K8A background (D). (E) HigB patch two residues (R29 and R69) were singly and in combination changed to alanine and their effect on E. coli growth was monitored at an OD at 600 nm over 6 h. For panels B–D, error bars display standard error of the mean from at least three experiments.
