Poster abstracts

Poster number 144 submitted by Lucia Beste Zehnder

Elucidation of RyhB target prioritization in E. coli

Lucia Beste Zehnder (Department of Biology, Gustavus Adolphus College), Ella Chuinard (Department of Biology, Gustavus Adolphus College), Geneva Fackler (Biochemistry and Molecular Biology Program, Gustavus Adolphus College), Marisen Turetzky (Biochemistry and Molecular Biology Program, Gustavus Adolphus College), Emma Zoeller (Biochemistry and Molecular Biology Program, Gustavus Adolphus College), Jane K Frandsen (Departments of Biology and Chemistry, Biochemistry and Molecular Biology Program, Gustavus Adolphus College)

Abstract:
Bacterial small RNAs (sRNAs) are non-coding RNAs that regulate gene expression by binding to mRNAs to repress or activate protein expression. sRNAs have a targetome of multiple mRNAs that they regulate. We hypothesized that these mRNA targets vary in priority; higher-priority targets are regulated when sRNA levels are low, while lower-priority targets are affected as sRNA levels increase. We aim to elucidate the hierarchy of the Escherichia coli RyhB targetome to better understand what dictates target prioritization. To investigate the regulatory hierarchy of the RyhB targetome, we are using two complementary assays. To assess the hierarchy of the binding interactions between RyhB and its mRNA targets, we have developed a pull-down assay by modifying the MS2 affinity purification coupled with RNA sequencing (MAPS) technique.1 Instead of using RNA sequencing at one time point, we use RT-qPCR to quantify the amounts of the RyhB mRNA targets pulled down over a time course after MS2-RyhB induction. To assess the hierarchy of the change in protein expression from mRNA targets, we are using a two-plasmid assay similar to one used previously to study SgrS.2 In this assay, RyhB and a target mRNA fused to GFP are expressed using independently controlled inducible promoters; in the presence of different combinations of inducers, we use a plate reader to monitor GFP fluorescence and bacterial growth over time. When comparing different mRNA targets, differences in GFP expression over the inducer range and time course suggest differences in the regulatory hierarchy. We inserted each RyhB mRNA target into the GFP fusion plasmid and began analyzing each target independently. After elucidating the hierarchy, we plan to assess mRNA target features, such as binding-site accessibility, to better understand the features that dictate prioritization and promote the dismantling of bacterial protein regulation in the fight against antibiotic resistance.

References:
1) Lalaouna, D., Carrier, M.-C., Semsey, S., Brouard, J.-S., Wang, J., Wade, J.T., and Massé, E. (2015).
A 3’ external transcribed spacer in a tRNA transcript acts as a sponge for small RNAs to prevent
transcriptional noise. Mol. Cell 58, 393–405. https://doi.org/10.1016/j.molcel.2015.03.013.
2) Bobrovskyy, M., Azam, M.S., Frandsen, J.K., Zhang, J., Poddar, A., Ma, X., Henkin, T.M., Ha, T.,
and Vanderpool, C.K. (2019). Determinants of target prioritization and regulatory hierarchy for the
bacterial small RNA SgrS. Mol. Microbiol. 112, 1199–1218. https://doi.org/10.1111/mmi.14355.

Keywords: small RNAs, gene regulation, prioritization