Poster abstracts
Poster number 34 submitted by Fawwaz Naeem
The anti-Shine-Dalgarno element of 16S rRNA is needed for translation of one gene, rpsU, in Flavobacterium johnsoniae
Fawwaz M. Naeem (Ohio State Biochemistry Program, Center for RNA Biology, OSU), Bappaditya Roy (Department of Microbiology, Center for RNA Biology, OSU), Dominic Arpin, Joaquin Ortega (Department of Anatomy and Cell Biology, McGill University), Zakkary A. McNutt (Ohio State Biochemistry Program, Center for RNA Biology, OSU), Kyung-Mee Moon, Leonard J. Foster (Department of Biochemistry and Molecular Biology, University of British Columbia), Kurt L. Fredrick (Department of Microbiology, Center for RNA Biology, OSU)
Abstract:
Bacteria of class Bacteroidia lack Shine-Dalgarno (SD) sequences in mRNA. Bacteroidia ribosomes are “blind” to SD sequences, both in vivo and in vitro, even though they contain the conserved anti-SD (ASD) element of 16S rRNA. 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. Interestingly, there is one gene of F. johnsoniae which 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. To investigate the broader role of the ASD in Bacteroidia, we systematically targeted the ASD of each 16S rRNA gene, substituting the core element at 4 of 5 positions (CCUCC to GAAGC). Consecutive replacement of each gene with this quadruple-substituted (QS) allele had little effect on cell growth until the last gene was changed. The final strain, containing only QS ribosomes, grows very slowly, with a doubling time >4-fold larger than wild-type. This growth defect can be largely rescued by rewiring translation of rpsU—i.e., replacing the native translation initiation region (TIR) with the SD-less TIR of the EF-Tu gene (tuf). Purified QS ribosomes are unable to translate native rpsU mRNA but are active on other mRNAs, in line with the genetic data. We also selected fast-growing suppressors of the ASD-ablated strain. Many of these suppressors carry single substitutions in the SD sequence of rpsU, pointing to the importance of bS21 production. Interestingly, wild-type ribosomes fail to initiate on these variant rpsU mRNAs, demonstrating that an extended SD-ASD duplex is normally needed for initiation. Finally, we found that ablation of the ASD inhibits rRNA processing during ribosome biogenesis. Overabundance of precursor (pre)-16S species correlated with the number of QS alleles but had no clear relation to growth rate. Collectively, these findings indicate that the main purpose of the ASD in F. johnsoniae is to facilitate translation of one gene, rpsU.
References:
Jha V, Roy B, Jahagirdar D, McNutt ZA, Shatoff EA, Boleratz BL, Watkins DE, 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. doi:10.1093/nar/gkaa1195
McNutt ZA, Roy B, Gemler BT, Shatoff EA, Moon K-M, Foster LJ, Bundschuh R, Fredrick K. 2023. Ribosomes lacking bS21 gain function to regulate protein synthesis in Flavobacterium johnsoniae. Nucleic Acids Res 51: 1927–1942. doi:10.1093/nar/gkad047
Keywords: Ribosomes, RNA , Translation autoregulation, Flavobacterium johnsoniae