RRM
2008 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
Class II prolyl-tRNA synthetase (ProRS) is a multidomain protein that attaches Pro to its cognate tRNAPro. In addition to an aminoacylation active site, most bacterial ProRSs also possess a separate editing domain (INS) that maintains fidelity of protein synthesis by hydrolyzing noncognate Ala mischarged onto tRNAPro. Escherichia coli (Ec) ProRS also misactivates Cys but is unable to hydrolyze Cys-tRNAPro via its INS editing domain. Instead, a small free-standing protein homologous to the INS domain (YbaK) has been shown to hydrolyze mischarged Cys-tRNAPro in trans, constituting a triple-sieve mechanism of editing. The gram-positive bacterium Enterococcus faecalis (Ef), which is an emerging pathogen in hospital-acquired infections, also encodes a YbaK homolog. Here, aminoacylation and hydrolytic editing activities of Ef ProRS and Ef YbaK are examined by performing cognate Pro charging, noncognate Cys mischarging, and Ala-, Ser- & Cys-tRNAPro deacylation assays. Ef ProRS possesses similar cognate Pro aminoacylation and noncognate Cys mischarging activity as Ec ProRS at 37 °C. Cys mischarging by Ef ProRS was abolished in the presence of Ef YbaK, consistent with the latter protein’s Cys-tRNAPro deacylase function. As expected, deacylation assays showed that Ef ProRS can hydrolyze mischarged Ala- but not Ser- or Cys-tRNAPro, while Ef YbaK can clear Cys-tRNAPro. In ongoing studies, the in vitro interaction between ProRS, YbaK and tRNAPro is being probed by fluorescence spectroscopy and the binding sites between them are being mapped by mass spectrometric protein footprinting. To gain further mechanistic insights into their biological roles, in vivo interactions of YbaK and other homologous proteins of unknown function, such as Ec YeaK, are being investigated using tandem affinity purification and mass spectrometry.
Keywords: ProRS, YbaK, Tandem affinity purification
