Poster abstracts

Poster number 87 submitted by Zakkary McNutt

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

Zakkary McNutt (Ohio State Biochemistry Program and Department of Microbiology), 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-SD (ASD) at the 3’ end of the 16S rRNA. Recent genomic studies have revealed that certain bacterial lineages, including those of the Bacteroidetes phylum, completely lack SD sequences [1]. Despite this, the conserved ASD is retained, suggesting an alternative function. 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, systematic mutagenesis of nucleotides 1534– 1542 of 16S rRNA suggests that five nucleotides (1535 CCUCC 1539) represent the functional core of the ASD. These mutant alleles 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. Three rrn operons have been deleted so far. This Δ3 strain exhibits a reduced growth rate, which can be partially complemented by a plasmid containing an rrn operon. In cells expressing heterogeneous 16S populations, mutant ribosomes can be tracked in sucrose gradients to assess changes in ribosome assembly and function. These experiments show that 30S subunits with mutations to the ASD largely retain function in F. johnsoniae, although these subunits are underrepresented in polysomes and accumulate in the 30S region of the gradient, indicating some defect in assembly or initiation.

References:
[1] 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