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Ribosomes of Bacteroidia fail to recognize Shine–Dalgarno (SD) sequences due to sequestration of the 3′ tail of the 16S rRNA on the 30S platform. Yet in these organisms, theprfBgene typically contains the programmed +1 frameshift site with its characteristic SD sequence. Here, we investigateprfBautoregulation inFlavobacterium johnsoniae, a member of the Bacteroidia. We find that the efficiency ofprfBframeshifting inF. johnsoniaeis low (∼7%) relative to that inEscherichia coli(∼50%). Mutation or truncation of bS21 inF. johnsoniaeincreases frameshifting substantially, suggesting that anti-SD (ASD) sequestration is responsible for the reduced efficiency. The frameshift site of certain Flavobacteriales, such asWinogradskyella psychrotolerans, has no SD. InF. johnsoniae, thisW. psychrotoleranssequence supports frameshifting as well as the native sequence, and mutation of bS21 causes no enhancement. These data suggest thatprfBframeshifting normally occurs without SD–ASD pairing, at least under optimal laboratory growth conditions. Chromosomal mutations that remove the frameshift or ablate the SD confer subtle growth defects in the presence of paraquat or streptomycin, respectively, indicating that both the autoregulatory mechanism and the SD element contribute toF. johnsoniaecell fitness. Analysis ofprfBframeshift sites across 2686 representative bacteria shows loss of the SD sequence in many clades, with no obvious relationship to genome-wide SD usage. These data reveal unexpected variation in the mechanism of frameshifting and identify another group of organisms, the Verrucomicrobiales, that globally lack SD sequences.
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Comparative Analysis of anti-Shine- Dalgarno Function in Flavobacterium johnsoniae and Escherichia coli
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.
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- Award ID(s):
- 2029502
- PAR ID:
- 10330715
- Date Published:
- Journal Name:
- Frontiers in Molecular Biosciences
- Volume:
- 8
- ISSN:
- 2296-889X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fmolb.2021.787388
