Poster abstracts

Poster number 44 submitted by Jo Marie Bacusmo

Probing the Function of the Truncated Insertion Domain in Rhodopsudomonas palustris Prolyl-tRNA Synthetase

Jo Marie Bacusmo (Departments of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Sandeep Kumar (Departments of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Karin Musier-Forsyth (Departments of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University, Columbus, OH 43210)

Abstract:
Aminoacyl-tRNA synthetases are responsible for charging the correct amino acid onto cognate tRNAs. To ensure fidelity in protein synthesis, a sub-set of these enzymes have evolved editing mechanisms. “Pre-transfer” editing refers to hydrolysis of the misactivated aminoacyl-adenylate intermediate, whereas “post-transfer” editing is hydrolysis of the mischarged tRNA. In most bacterial prolyl-tRNA synthetase (ProRS) systems, an editing domain known as INS is responsible for post-transfer editing of mis-charged Ala-tRNAPro. However, a sub-set of ProRSs have a truncated “mini–INS” that is unlikely to support catalytic activity. To explore the function of the mini-INS and to better understand why a full-length INS is not needed in some bacteria, we chose to investigate Rhodopseudmonas palustris (Rp) ProRS. The mini-INS consists of 28 residues forming 2 α-helices and a loop. We showed that RpProRS activates Cys, Pro, and Ala with 0-, 5-, and 25-fold reduced catalytic efficiency relative to EcProRS. The estimated KM of 1.0 M for Ala is significantly higher than the concentration of Ala in the cell. In addition, RpProRS shows pre-transfer activity against Ala that is comparable to that of EcProRS. Therefore, based on the lower overall activation of Ala and the relatively robust pre-transfer editing activity, we conclude that a full-length INS domain for post-transfer editing is not needed in the Rp system.
Based on the known dimeric structure of RpProRS and preliminary gel filtration studies, we hypothesize that residues in the mini-INS domain have been maintained to facilitate tight dimer formation. Modeling studies suggest that acidic residues on the loop in the mini-INS domain interact with basic residues proximal to the known dimerization interface. We hypothesize that this salt bridge aids in the oligomerization process. In support of this hypothesis, all mini-INS deletion variants prepared to date were found to be misfolded and formed inclusion bodies. Taken together, our results suggest that the mini-INS is critical for preserving RpProRS’s active dimeric structure.

Keywords: Rhodopseudomonas palustris, aminoacyl-tRNA synthetases, post-transfer editing