2009 Rustbelt RNA Meeting
RRM

 

Registration

Agenda

Abstracts

Directions

Talk abstracts

Talk on Friday 12:55-01:10pm submitted by Mom Das

Identification of New Partners for Bacterial YbaK Protein In Vivo: Implications for Translational Quality Control

Mom Das (Ohio State Biochemistry Program, The Ohio State University), Byung Ran So (Department of Chemistry, The Ohio State University), Mohosin Sarkar (Department of Chemistry, The Ohio State University), Thomas J. Magliery (Departments of Chemistry and Biochemistry, The Ohio State University), Karin Musier-Forsyth (Departments of Chemistry and Biochemistry, The Ohio State University)

Abstract:
Prolyl-tRNA synthetase (ProRS) is a multi-domain protein that specifically ligates Pro to its cognate tRNAPro in a reaction catalyzed by its aminoacylation active site. Most bacterial ProRSs also possess an editing domain (INS), a discrete active site that functions to hydrolyze noncognate Ala mischarged onto tRNAPro. In addition, Escherichia coli (Ec) ProRS misactivates Cys but is unable to hydrolyze Cys-tRNAPro via its INS domain. Instead, a small free-standing protein homologous to the INS domain (YbaK) has been shown to hydrolyze mischarged Cys-tRNAPro in trans. YbaK behaves as a general Cys-deacylase in vitro, hydrolyzing all Cys-tRNA substrates independent of tRNA identity. This calls into question how YbaK gains specificity for Cys-tRNAPro and whether there are any other mischarged Cys-tRNA substrates for YbaK in vivo. To gain further mechanistic insights into the biological roles of YbaK, in vivo interactions were probed using a tandem affinity purification (TAP-tag) method to identify proteins interacting with YbaK. Interestingly, in addition to ProRS, three additional synthetases, AlaRS, LysRS and AsnRS, were identified as interacting partners of YbaK when a formaldehyde cross-linking step was included. To further validate these interactions, a split-GFP reassembly technique was used to probe these protein-protein interactions in vivo. We also probed the possible functional implications of these interactions by performing deacylation assays by YbaK in the presence and absence of the interacting partners. Taken together, we propose that YbaK may function in vivo to hydrolyze Cys-tRNA species other than Cys-tRNAPro, and that interactions with the corresponding synthetases may play a role in substrate channeling and translational quality control.

Keywords: ProRS, YbaK, TAP-tag, Split GFP technique