Poster abstracts
Poster number 29 submitted by Fawwaz Naeem
Reduced efficiency of prfB programmed frameshifting in Flavobacterium johnsoniae due to ASD sequestration
Fawwaz Naeem (OSBP, The Ohio State University), Zakkary A. McNutt (OSBP, The Ohio State University), Kurt Fredrik (Microbiology Department, The Ohio State University)
Abstract:
In bacteria, two release factors are responsible for translation termination—RF1 and RF2. Production of RF2 is autoregulated in most bacteria (> 80%) via programmed frameshifting. In short, this entails a competition between RF2-dependent termination at an in-frame UGA codon and +1 frameshifting of peptidyl-tRNALeu on the slippery sequence CUUU. The frameshifting event is promoted by a SD-like sequence closely juxtaposed to the slippery sequence. Ribosomes of Bacteroidia fail to recognize SD sequences due to sequestration of the 3’ tail of the 16S rRNA on the 30S platform. Yet, in these organisms, the prfB gene typically contains the frameshift site with its characteristic SD-like sequence. Here we investigate prfB autoregulation in F. johnsoniae, a member of the Bacteroidia. We show that the efficiency of prfB frameshifting in F. johnsoniae is low (5-10%) relative to that in E. coli (~50%). Mutation or truncation of bS21 in F. johnsoniae increases frameshifting substantially, suggesting that ASD sequestration is responsible for the reduced efficiency. The frameshift site of certain Flavobacteriales, such as Winogradskyella psychrotolerans, has lost the SD-like sequence. In F. johnsoniae, this W. psychrotolerans sequence supports frameshifting at 5-10%, which is unaffected by mutation of bS21. Collectively, these data suggest that prfB frameshifting is largely independent of SD-ASD pairing in the Bacteroidia and consequently tuned down.
References:
1. Baez WD, Roy B, McNutt ZA, Shatoff EA, Chen S, Bundschuh R, Fredrick K. Global analysis of protein synthesis in Flavobacterium johnsoniae reveals the use of Kozak-like sequences in diverse bacteria. Nucleic acids research. 2019;47(20):10477-10488. doi:10.1093/nar/gkz855
2. Jha V, Roy B, Jahagirdar D, McNutt ZA, Shatoff EA, Boleratz BL, Watkins DE, Bundschuh R, Basu K, Ortega J, Fredrick K. Structural basis of sequestration of the anti-Shine-Dalgarno sequence in the Bacteroidetes ribosome. Nucleic Acids Research. 2021;49(1):547-567. doi:10.1093/nar/gkaa1195
3. Weiss RB, Dunn DM, Atkins JF, Gesteland RF. Slippery runs, shifty stops, backward steps, and forward hops: -2, -1, +1, +2, +5, and +6 ribosomal frameshifting. Cold Spring Harbor Symposia on Quantitative Biology. 1987;52: 67-693. doi:10.1101/SQB.1987.052.01.078
Keywords: RF2 (prfB) autoregulation, Ribosome , Bacteroidia