Figure 2. Sites and sequences used in rrs engineering.

(A) Secondary structure of the M. mycoides 16S rRNA in which the six locations where the gene was engineered and the seven mutations incorporated from the M. capricolum rrs are indicated. rrs carrying M. capricolum mutations was used as a template to generate the single-, dual- or triple-site mutations. (B) Sites where of insertion elements were added to the M. mycoides 16S rRNA. Positions at which each of the six helices were “opened” to insert heterologous elements are shown using red lines. (C) Insertion elements used in rrs engineering. Sequence of the MS2-coat protein binding RNA (MS2), the catalytically-inactive hammer-head ribozyme (HHRi) and the synthetic, scar helices (SH) are shown. Sequences of the MS2 and HHRi elements added to each of the six sites shown in (A) are the same while the SH element added to each one of six sites varied in the sequences flanking the common hairpin structure. MS2 element was flanked by SpeI sites (green) as described in Youngman et al.³⁴. G→A mutation that renders the hammer-head ribozyme catalytically-inactive is indicated. (D) Helix substitutions. Four helices, h6, h10, h17 and h39 from M. mycoides were substituted with those from B. subtilis, C. difficile and E. coli. Sequences and secondary structures of the wild-type and the heterologous helices that were used for substitutions are shown.