Poster abstracts
Poster number 138 submitted by Annie Urban
Investigating the role of bS21 paralogs in ribosome heterogeneity in Agrobacterium fabrum
Annie Urban (Ohio State University), Fawwaz Naeem (Ohio State University), Kurt Fredrick (Ohio State University)
Abstract:
The ribosome is the machine responsible for translation of mRNAs into proteins needed for cellular functions. In bacteria, ribosomes are made up of two subunits, small 30S and large 50S. The 30S subunit contains the 16S rRNA which has the anti-Shine Dalgarno (ASD) sequence located on its 3`end. Typically, during translation initiation, the ASD base pairs with the Shine-Dalgarno (SD) sequence located upstream of the start codon on mRNAs and this SD-ASD interaction positions the start codon in the P-site. Located near the ASD is the ribosomal protein bS21 which help stabilize the ASD-SD binding. bS21 is essential in some bacteria while completely absent from others, and some bacteria have multiple copies. Recently, it has been shown that bS21 is involved in regulation of its own synthesis in Flavobacterium johnsoniae. Furthermore, bS21 is also a common ribosomal protein encoded by phages. It has been shown to get incorporated into the ribosome during late-stage infection and may explain how viruses replicate using host machinery.
In Agrobacterium fabrum, there are three different rpsU (bS21) genes: ATU4064, ATU3123, and ATU3637, denoted as rpsU-A, rpsU-B, and rpsU-C respectively. The genes are identical except in the C-terminal, suggesting duplications or redundancy functions; however, preliminary data suggests these homologs can act as global regulators of translation. RNA-seq studies show differential expression of the three genes in different media. In rich media, rpsU-A was more highly expressed; however, in minimal media, rpsU-B, and rpsU-C are more expressed. This suggests that there could be heterogenous populations of ribosomes within the cell as a funcntion of which bS21 homolog is incorporated into the ribosomes, which could serve as a stress response to changing nutrient conditions. We have generated multiple deletion strains to investigate the role of each of the three native A. fabrum genes along with phage mutants that only contain phage encoded S21 proteins from three different phages. These phage genes are identical to the native A. fabrum genes except for a variable and extended C-terminal region. Growth assays from these strains show a surprising phenotype where the rpsU-C deletion grows faster in minimal media, while the rpsU-B deletion is slower in rich media. Strains in which phage genes were inserted into A. fabrum genome show a defect in growth. Currently, we are performing Ribo-Seq on those various strains to elucidate the role of bS21 paralogs in ribosome heterogeneity and investigate the possibility of bS21 phage genes being used to redirect translation of the phage machinery in the host.
Keywords: RNA, Ribosome heterogeneity, Phages