in vivo function
Our mission is to identify the mechanistic basis of bacterial translation and regulation through a combination of biochemical, structural, and molecular biology techniques.
Translational recoding and miscoding
The ribosome reads genetic information encoded on the mRNA template and translates this into amino acids that are the basis of all cellular proteins. How does the ribosome maintain its canonical three-nucleotide mRNA frame, and how does miscoding occur?
We use frameshift suppressor tRNAs that alone cause a mRNA frameshift (decode a non-three-nucleotide codon to return the ribosome to the correct reading frame) (Fagan et al., RNA 2014; Maehigashi et al., PNAS 2014; Hong et al., PNAS 2018), modified tRNAs implicated in mRNA frame maintenance (Nguyen et al. JBC 2019 Hoffer et al., eLife 2020; Nguyen et al., PNAS 2020), and 16S rRNA ribosome ambiguity mutations (ram), a hyperaccurate phenotype (Fagan et al., PNAS 2013; Hoffer et al., NAR 2018), to identify ribosomal changes and establish frameshifting and miscoding event timings.
Bacterial ribosome rescue as a novel antimicrobial pathway
Bacteria use a mechanism unique from eukaryotes to rescue stalled ribosomes (trans-translation) that has been established as a novel antibiotic pathway with several hit candidates (Ramadoss et al., PNAS 2013). Pharmacokinetically active derivatives were synthesized with high efficacy in a mouse model (Aron et al., Nat Comm 2021). We established the novel binding site of these drugs and show that there is a dramatic movement of the ribosomal protein bL27 N-terminus in response to drug binding, indicating a novel mode of action.
Bacterial toxin activation and regulation.
We are interested in how protein synthesis is halted during environmental cures through toxin-antitoxin regulation, allowing bacteria to enter an antibiotic-tolerant state, Type II toxin-antitoxin systems are abundant, beneficial gene pairs where the toxin protein functions as a ribonuclease and targets important RNAs including mRNAs, tRNAs, and rRNAs (Schureck et al., JBC 2014; Ruangprasert et al., JBC 2014; Maehigashi et al., NAR 2015; Schureck et al., PNAS 2015; Schureck et al., RNA 2016; Schureck et al., NAR 2016; Hoffer et al., JBC 2017; Pavelich et al., NAR 2019). We have identified specificity of toxin proteins for mRNAs undergoing protein synthesis and want to learn how antitoxins are selectively degraded within bacteria so toxins may carry out their functions.