Poster abstracts

Poster number 57 submitted by Dawt Sang

Creating Chimeric tRNAs to Study Substrate Recognition by a Trans-Editing Enzyme

Dawt Sang (Department of Biology, Department of Chemistry, Earlham College), Vy Nguyen (Department of Biology, Department of Chemistry, Earlham College), Landry Cox (Department of Biology, Department of Chemistry, Earlham College), Samia Kassim (Department of Biology, Department of Chemistry, Earlham College), Lexie Kuzmishin Nagy (Department of Biology, Department of Chemistry, Earlham College)

Abstract:
Aminoacyl-tRNA synthetases (aaRSs) pair an amino acid to its corresponding tRNA.1 However, due to structural similarities, aaRSs can mischarge amino acids, including D-amino acids, onto tRNAs. Frequent D-amino acid mischarging threatens cells as it reduces the pool of tRNA available for translation. As a result, some organisms have evolved post-transfer editing domains to correct these errors.2 ProXp-x is a single-domain editing enzyme found in R. palustris and other rhizobia that can edit tRNAs mischarged with D- and L-amino acids. Thus, ProXp-x may protect the proteome and cell from proteinogenic amino acids. Previous data show that ProXp-x deacylates alpha-aminobutyrate (Abu) mischarged onto tRNAVal and tRNAPro at faster rates than L-Ala.3 Additionally, Abu-tRNAVal deacylation is faster than Abu-tRNAPro, suggesting tRNA identity impacts substrate selection and rate. Furthermore, ProXp-x deacylates D-Ala-tRNAPro, but not D-Ala-tRNAAla, suggesting tRNA identity may even influence editing of the amino acid enantiomer. We investigated the tRNA identity elements that ProXp-x recognizes for substrate selection and deacylation. We induced mutations in select tRNAs via site directed mutagenesis. We’re in the process of using isothermal titration calorimetry4, DRaCALA assays5, and deacylation assays6 with the mutated tRNAs and WT tRNAs to measure binding affinity and determine acceptor stem identity elements that dictate substrate recognition. We also confirmed the successful deletion/presence of the D-aminoacyl-tRNA deacylase gene (dtd) in E. coli WT and &deltadtd strains.7 These strains will be used in future experiments to test ProXp-x’s ability to rescue E. coli from D-amino acid toxicity. PCR and sequencing results verified the WT and deletion strains.

References:
1. Ibba, M. & Söll, D. (2000). Annual Review of Biochemistry, 69, 617–650. https://doi.org/10.1146/annurev.biochem.69.1.617.

2.Hussain, T., et al. (2006). The EMBO Journal, 25(17), 4152–4162. https://doi.org/10.1038/sj.emboj.7601278.

3. Bacusmo, J. M. & Kuzmishin, A. B. et al. RNA Biology, 15(4–5), 576–585. https://doi.org/10.1080/15476286.2017.1353846.

4. Seminara, A. B., et al. (2019). Current Protocols in Molecular Biology, 126, e84. https://doi.org/10.1002/cpmb.84.

5. Salim, N. N., & Feig, A. L. (2009). Methods, 47(3), 198–205. https://doi.org/10.1016/j.ymeth.2008.09.003.

6. Splan, K. E., et al. (2008). Methods, 44(2), 119–128. https://doi.org/10.1016/j.ymeth.2007.10.009.

7. Baba, T., et al. (2006). Molecular Systems Biology, 2, 2006.0008. https://doi.org/10.1038/msb4100050.

Keywords: tRNA, editing, D-amino acids