Poster abstracts
Poster number 107 submitted by Fawwaz Naeem
Why Bacteroidia ribosomes tuck in their tails
Fawwaz M. Naeem (Ohio State Biochemistry Program, Center for RNA Biology, OSU), Bappaditya Roy (Center for RNA Biology, Department of Microbiology, OSU), Kurt Fredrick (Ohio State Biochemistry Program, Center for RNA Biology, Department of Microbiology, OSU)
Abstract:
Certain groups of bacteria, including the Bacteroidia, generally lack Shine-Dalgarno (SD) sequences. Bacteroidia ribosomes contain the anti-Shine-Dalgarno (ASD) element but fail to recognize SD sequences, both in vivo and in vitro. A cryo-EM structure of the Flavobacterium johnsoniae ribosome shows that the 3’ tail of 16S rRNA is sequestered in a pocket formed by bS21, bS18, and bS6 on the 30S platform, explaining the basis of ASD inhibition (1). Interestingly, one gene in F. johnsoniae contains a strong SD sequence—rpsU, which encodes bS21. F. johnsoniae ribosomes lacking bS21 exhibit a liberated ASD and translate rpsU at a higher rate, which creates a feedback loop that autoregulates bS21 production (2). The platform pocket is conserved across members of Bacteroidia, even those with no apparent bS21 autoregulation, hinting at a broader role for ASD sequestration. Recently, we have systematically targeted the ASD of each 16S rRNA gene in F. johnsoniae, substituting the core element at 4 of 5 positions (CCUCC to GAAGC). This quadruple mutation (QM) should not only abolish SD recognition but also liberate the 3’ tail from the pocket. Consecutive replacement of each 16S gene with the QM allele had little effect on cell growth until the last gene was changed. The final strain, containing only QM ribosomes, grows very slowly, with a doubling time >4-fold larger than wild-type. This growth defect can be largely rescued by a plasmid-borne copy of rpsU with the SD-less leader of the EF-Tu gene (tuf). This underscores the importance of SD-ASD pairing for translation of endogenous rpsU in F. johnsoniae. However, re-wiring of rpsU translation cannot fully restore growth rate, indicating that the QM ribosomes are compromised in some other way. Currently, we are studying mutations in bS18 and bS6 that specifically target the platform pocket. Our initial findings suggest that initiation on SD-less mRNA depends on the integrity of the pocket. Thus, ASD sequestration appears to play both regulatory and housekeeping roles in the Bacteroidia.
References:
1. Jha, V., Roy, B., Jahagirdar, D., McNutt, Z.A., Shatoff, E.A., Boleratz, B.L., Watkins, D.E., Bundschuh, R., Basu, K., Ortega, J. et al. (2021) Structural basis of sequestration of the anti-Shine-Dalgarno sequence in the Bacteroidetes ribosome. Nucleic Acids Res, 49, 547-567.
2. McNutt, Z.A., Roy, B., Gemler, B.T., Shatoff, E.A., Moon, K.M., Foster, L.J., Bundschuh, R. and Fredrick, K. (2023) Ribosomes lacking bS21 gain function to regulate protein synthesis in Flavobacterium johnsoniae. Nucleic Acids Res, 51, 1927-1942.
Keywords: Ribosome, Flavobacterium johnsoniae , bS21