Poster abstracts

Poster number 41 submitted by Zakkary McNutt

Investigating the role of the anti-Shine Dalgarno in the Bacteroidetes

Zakkary McNutt (Ohio State Biochemistry Program), Mai Dang (Ohio State University Department of Microbiology), Bappaditya Roy (Ohio State University Department of Microbiology), Aishwarya Devarag (Ohio State University Department of Microbiology), Kurt Fredrick (Ohio State University Department of Microbiology)

Abstract:
Translation initiation in bacteria often entails pairing between the Shine Dalgarno (SD) sequence of mRNA and the anti-Shine Dalgarno (ASD) at the 3’ end of the 16S rRNA. Genomic studies have revealed that certain bacterial lineages, such as the Bacteroidetes, completely lack SD sequences [Nakagawa et al., 2010]. Despite this, the ASD remains conserved in these organisms, implying an alternative function of the ASD. Here, we present our initial work to probe the function of the ASD in Flavobacterium johnsoniae, a genetically tractable member of the Bacteroidetes. In E. coli, ribosomes harboring substitutions in the core ASD (CCUCC) fail to support cell growth and cause dominant lethality. We have begun to generate and characterize analogous mutations in the 16S rRNA of F. johnsoniae. This organism contains six virtually identical rrn operons. We are working to delete all 6 operons from the chromosome, such that cells are supported by a single plasmid-born copy of the rrn operon, akin to the Δ7 prrn strain of E. coli. Three rrn operons have been deleted so far. This Δ3 strain exhibits a reduced growth rate, which can be complemented by a plasmid containing the rrn operon. Mutant rrn alleles, with up to 4 substitutions in the ASD, complement the Δ3 strain to various degrees. In cells expressing heterogeneous 16S populations, polysome analysis revealed that though still functional, mutant rrn alleles accumulate in earlier sucrose fractions.

References:
Nakagawa, S., Niimura, Y., Miura, K. & Gojobori, T. Dynamic evolution of translation initiation mechanisms in prokaryotes. Proc Natl Acad Sci USA 107, 6382-6387, doi:10.1073/pnas.1002036107 (2010).

Keywords: Ribosome, RNA, Shine Dalgarno