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. 2010 Feb 22;107(10):4567–4572. doi: 10.1073/pnas.0912305107

Fig. 4.

Fig. 4.

Structural considerations for the leader sequence of 16S rRNA and translational fidelity. (A) Secondary structure of E. coli 16S rRNA (40) with the region containing the mature 5′ end and the central pseudoknot in box. Two sets (I and II) of possible base-pairing configurations are shown in the box to form alternative helices #1 and #2 (see text). Mature 16S rRNA is shown in blue and leader sequence is shown in brown. Helices 1 and 2 as observed in mature 16S rRNA [derived from the crystal structure of 70S ribosome of E. coli (41)] are shown in equilibrium with leader specific helices (prehelices) which precludes the formation of helix 1 and may hinder formation of helix 2 (see ref. 14). Nucleotides in parenthesis in panel II are absent in lp16S rRNA (see text), but present in the primary transcript. Solid lines denote Watson–Crick bonding, whereas solid circles denote canonical non-Watson–Crick interactions (40) and empty circles denote additional interactions based on the X-ray crystal structure of E. coli 70S ribosome 41. (B) Primer extension analysis of RNA extracted from SSUs and 70S ribosomes modified with DMS and isolated from S5(G28D), ΔrpsO, and Δrng strains (and their parental strains). A and G correspond to the dideoxy sequencing lanes. Two control lanes are unmodified W3110 SSUs and 70S ribosomes. Lanes 1–12 are modified by DMS (see Methods). Lanes 1, 2, 5, 6, 9, and 10 are SSUs and lanes 3, 4, 7, 8, 11, and 12 are 70S ribosomes from CSH142 (lanes 1 and 3), S5(G28D) (lanes 2 and 4), W3110 (lanes 5 and 7), ΔrpsO (lanes 8 and 10), BW25113 (lanes 9 and 11), or Δrng (lanes 10 and 12). Altered reactivity in S5(G28D), ΔrpsO, and Δrng mutant particles relative to parental particles is marked with a solid box-headed arrow. As shown, 70S ribosomes from the parental strains lack p16S rRNAs and thus do not extend beyond the mature 5′ end. (C) Structural changes in positions A7 and A8 of 16S as indicated by the solid box-headed arrow in B are quantified using ImageJ (National Institutes of Health). Protections in S5(G28D) (brown bars), ΔrpsO (green bars), and Δrng (purple bars) are denoted as percentages of their parental strains for both the SSU and 70S ribosomes.