Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Poster Abstracts

50-POS Board 50 APTAMER-MEDIATED INHIBITION OF BACTERIAL INITIATION FACTOR IF3 Ana E Sanchez-Castro 1 ; Katherin Peñaranda 1 ; Daria S Vinogradova 2 ; Andrey L Konevega 2 ; Pohl Milón 1 ; 1 Universidad Peruana de Ciencias Aplicadas (UPC), Health Sciences Faculty, Lima 15023, Peru 2 Petersburg Nuclear Physics Institute , Gatchina 188300, Russian Federation Bacterial translation initiation factor IF3 is an essential protein that promotes 30S initiation complex (IC) formation. IF3 binds the 30S ribosomal subunit and modulates the fidelity and speed of the codon-anticodon interaction between the mRNA and initiator tRNA. Although IF3 functions are well understood, the role of the N terminal domain (IF3N) of the factor remains debated. Here, we use Systematic Evolution of Ligands by EXponential enrichment (SELEX) to develop aptamers for IF3N and biophysical methods to study aptamer-mediated inhibition of 30S IC assembly. Purified IF3N from Escherichia coli was used as target for aptamers selection. Five potential aptamers were identified and chemically synthesized. Pull-down assays using target- immobilized magnetic particles suggested four molecules as potential binders. Three aptamers showed dissociation constants in the range of 100-400 nM, as assayed by Microscale Thermophoresis (MST). Intramolecular Förster Resonance Energy Transfer (FRET) showed that all three aptamers are able to bind a double labeled IF3; however, only one molecule was able to drastically reduce the speed of IF3 binding to the 30S subunit. The aptamer affected IF1- and IF2-depended conformational accommodations of IF3 on the 30S subunit. Furthermore, the canonical codon-anticodon association appeared to be affected by the aptamer as assayed by rapid kinetics and MST. Altogether, our results suggest that IF3N positioning contributes with the accommodation of initiator tRNA and therefore, with canonical 30S IC formation. Additionally, the experimental scheme presented here provides a solid alternative for the development of new inhibitors of bacterial translation.

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