Poster abstracts

Poster number 6 submitted by Mom Das

Role of Bacterial ProRS Editing Domain Homologs in Translational Quality Control

Mom Das (Department of Chemistry, The Ohio State University), Karin Musier-Forsyth (Departments of Chemistry and Biochemistry, The Ohio State University)

Abstract:
Class II prolyl-tRNA synthetase (ProRS) is a multidomain protein that specifically attaches Pro to its cognate tRNA^Pro via an aminoacylation active site. Most bacterial ProRSs also possess an editing domain (INS), a discrete active site that functions to hydrolyze noncognate Ala mischarged onto tRNA^Pro. In addition to Ala misactivation, Escherichia coli (Ec) ProRS misactivates Cys but is unable to hydrolyze Cys-tRNA^Pro via its INS editing domain. Instead, a small free-standing protein homologous to the INS domain (YbaK) has been shown to hydrolyze mischarged Cys-tRNA^Pro in trans, constituting a triple-sieve mechanism of editing. However, the mechanism of specific substrate selection by this single domain hydrolase is unknown. YbaK alone appears to lack specific tRNA recognition capabilities and it behaves as a general Cys-deacylase by hydrolyzing both Cys-tRNA^Pro and Cys-tRNA^Cys in vitro. Here we demonstrate that Cys-tRNA^Pro deacylation activity of YbaK is enhanced in complex with Ec ProRS in vitro. This functional data together with earlier in vitro binding studies provide strong support for the hypothesis that the specificity of trans-editing by YbaK is ensured through formation of a novel ProRS/YbaK/tRNA complex. In this work, we aim to precisely map the sites of interaction between Ec ProRS and YbaK using a mass-spectrometric protein-footprinting approach. 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 also being investigated using tandem affinity purification and mass spectrometry. This method will help in identifying proteins interacting with YbaK or YeaK in stable complexes, as well as more transiently in non-stoichiometric amounts.

Keywords: ProRS, YbaK, YeaK, tandem affinity purification