Role of the anti-Shine-Dalgarno sequence of 16S rRNA in Flavobacterium johnsoniae

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 Flavobacterium johnsoniae, one gene contains a strong SD sequence-rpsU, which encodes bS21. Flavobacterium johnsoniae ribosomes lacking bS21 translate rpsU at a higher rate, which establishes an autoregulatory cycle. Here, we targeted the ASD of 16S rRNA in F. johnsoniae, 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 rpsU with the SD-less TIR of tuf. Purified QS ribosomes fail to translate native rpsU 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 rpsU. 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.