<span class="paragraphSection"><div class="boxTitle">Abstract</div>Bacteroidia ribosomes are “blind” to Shine–Dalgarno (SD) sequences because the anti-SD (ASD) of 16S rRNA is sequestered by bS21, bS18, and bS6 on the 30S platform. In <span style="font-style: italic;">Flavobacterium johnsoniae</span>, one gene contains a strong SD sequence—<span style="font-style: italic;">rpsU</span>, which encodes bS21. <span style="font-style: italic;">Flavobacterium johnsoniae</span> ribosomes lacking bS21 translate <span style="font-style: italic;">rpsU</span> at a higher rate, which establishes an autoregulatory cycle. Here, we targeted the ASD of 16S rRNA in <span style="font-style: italic;">F. johnsoniae</span>, ablating the core element (CCUCC to GAAGC). Replacement of each 16S gene with this quadruple-substituted (QS) allele had little effect until the last gene was changed. The final strain, containing only QS ribosomes, grows poorly. This defect can be largely rescued by replacing the translation initiation region (TIR) of <span style="font-style: italic;">rpsU</span> with the SD-less TIR of <span style="font-style: italic;">tuf</span>. Purified QS ribosomes fail to translate native <span style="font-style: italic;">rpsU</span> messenger RNA (mRNA) but are active on SD-less mRNAs. We also selected suppressors of the ASD-ablated strain, many of which carried a point mutation in the SD of <span style="font-style: italic;">rpsU</span>. Such mutations increase bS21 synthesis in the ASD-ablated strain but reduce bS21 synthesis by wild-type ribosomes, underscoring the importance of SD–ASD pairing in the natural case. We also find that ASD ablation inhibits rRNA processing, an effect independent of bS21.</span>