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Ribosomes of Bacteroidia (formerly Bacteroidetes) fail to recognize Shine-Dalgarno (SD) sequences even though they harbor the anti-SD (ASD) of 16S rRNA. Inhibition of SD-ASD pairing is due to sequestration of the 3’ tail of 16S rRNA in a pocket formed by bS21, bS18, and bS6 on the 30S platform. Interestingly, in many Flavobacteriales, the gene encoding bS21, rpsU, contains an extended SD sequence. In this work, we present genetic and biochemical evidence that bS21 synthesis in Flavobacterium johnsoniae is autoregulated via a subpopulation of ribosomes that specifically lack bS21. Mutation or depletion of bS21 in the cell increases translation of reporters with strong SD sequences, such as rpsU’-gfp, but has no effect on other reporters. Purified ribosomes lacking bS21 (or its C-terminal region) exhibit higher rates of initiation on rpsU mRNA and lower rates of initiation on other (SD-less) mRNAs than control ribosomes. The mechanism of autoregulation depends on extensive pairing between mRNA and 16S rRNA, and exceptionally strong SD sequences, with predicted pairing free energies of < –13 kcal/mol, are characteristic of rpsU across the Bacteroidota. This work uncovers a clear example of specialized ribosomes in bacteria.

 

The anti-Shine-Dalgarno (ASD) sequence of 16S rRNA is highly conserved across Bacteria, and yet usage of Shine-Dalgarno (SD) sequences in mRNA varies dramatically, depending on the lineage. Here, we compared the effects of ASD mutagenesis in Escherichia coli , a Gammaproteobacteria which commonly employs SD sequences, and Flavobacterium johnsoniae , a Bacteroidia which rarely does. In E. coli , 30S subunits carrying any single substitution at positions 1,535–1,539 confer dominant negative phenotypes, whereas subunits with mutations at positions 1,540–1,542 are sufficient to support cell growth. These data suggest that CCUCC (1,535–1,539) represents the functional core of the element in E. coli . In F. johnsoniae , deletion of three ribosomal RNA ( rrn ) operons slowed growth substantially, a phenotype largely rescued by a plasmid-borne copy of the rrn operon. Using this complementation system, we found that subunits with single mutations at positions 1,535–1,537 are as active as control subunits, in sharp contrast to the E. coli results. Moreover, subunits with quadruple substitution or complete replacement of the ASD retain substantial, albeit reduced, activity. Sedimentation analysis revealed that these mutant subunits are overrepresented in the subunit fractions and underrepresented in polysome fractions, suggesting some defect in 30S biogenesis and/or translation initiation. Nonetheless, our collective data indicate that the ASD plays a much smaller role in F. johnsoniae than in E. coli , consistent with SD usage in the two organisms. 

Abstract Genomic studies have indicated that certain bacterial lineages such as the Bacteroidetes lack Shine-Dalgarno (SD) sequences, and yet with few exceptions ribosomes of these organisms carry the canonical anti-SD (ASD) sequence. Here, we show that ribosomes purified from Flavobacterium johnsoniae, a representative of the Bacteroidetes, fail to recognize the SD sequence of mRNA in vitro. A cryo-electron microscopy structure of the complete 70S ribosome from F. johnsoniae at 2.8 Å resolution reveals that the ASD is sequestered by ribosomal proteins bS21, bS18 and bS6, explaining the basis of ASD inhibition. The structure also uncovers a novel ribosomal protein—bL38. Remarkably, in F. johnsoniae and many other Flavobacteriia, the gene encoding bS21 contains a strong SD, unlike virtually all other genes. A subset of Flavobacteriia have an alternative ASD, and in these organisms the fully complementary sequence lies upstream of the bS21 gene, indicative of natural covariation. In other Bacteroidetes classes, strong SDs are frequently found upstream of the genes for bS21 and/or bS18. We propose that these SDs are used as regulatory elements, enabling bS21 and bS18 to translationally control their own production. 

Viruses of two candidate phyla are abundant in the ocean and revise our understanding of early RNA virus evolution.