Fret-Based Approach to Probe Domain Motions upon ProRS/YbaK/tRNA_Pro Ternary Complex Formation

Abstract

To obtain a high level of accuracy during protein synthesis, several different quality control steps are employed by the cellular machinery. The aminoacyl-tRNA synthetases (aaRS) play a critical role in identifying amino acids and pairing them with their cognate tRNAs. Prolyl-tRNA synthetase (ProRS) from all three domains of life has been shown to mischarge alanine and cysteine onto tRNAPro. Most bacterial ProRSs have an editing domain that deacylates mischarged Ala-tRNAPro. However, this double-sieve editing mechanism is not sufficient to clear Cys-tRNAPro. Instead, a free-standing homolog of the ProRS editing domain called YbaK deacylates mischarged Cys-tRNAPro species.We have demonstrated that tRNAPro, ProRS and YbaK form a ternary complex in vitro and in vivo, but the details of this complex are not known.Based on preliminary computational studies, we hypothesize that the alanine editing domain of ProRS undergoes a conformational change to facilitate YbaK binding. In addition, the CCA-3′ end of the tRNA must also be involved in significant conformational changes, translocating between the synthetic active site, the ProRS editing domain active site located 35 A away, and YbaK. To probe these protein and RNA domain movements, we devised a fluorescence resonance energy transfer (FRET)-based approach. To date, using ensemble time-resolved FRET, we have measured an ∼20 A conformational change in the ProRS editing domain upon YbaK binding, confirming our hypothesis of a large conformational change to accommodate YbaK. Moreover, the distance between tRNA and YbaK differ by ∼15 A in the presence and absence of ProRS, further verifying a conformational change. Current studies are aimed at obtaining additional distance constraints between components of the ternary complex.