Poster abstracts

Poster number 38 submitted by Jo Marie Bacusmo

Fidelity of proline codon translation in Rhodopseudomonas palustris is maintained via a novel triple sieve mechanism of editing

Jo Marie Bacusmo (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University)

Abstract:
Aminoacyl-tRNA synethetases activate specific amino acids and transfer them to cognate tRNA isoacceptors. These enzymes are error prone due to misactivation of smaller or isosteric amino acids. In the case of prolyl-tRNA synthetase (ProRS), in addition to activating cognate Pro, Ala and Cys are readily misacylated. Thus, editing mechanisms have evolved to ensure fidelity of Pro codon translation. In many bacterial systems, a triple-sieve editing mechanism is employed, which consists of the ProRS active site that discriminates amino acids based largely on volume and size, the ProRS editing domain (INS) that hydrolyzes Ala-tRNAPro and also functions via size exclusion, and a free-standing INS homolog called YbaK, which clears Cys-tRNAPro via unique sulfhydryl side-chain chemistry. The gram-negative purple non-sulfur bacterium Rhodopseudomonas palustris (Rp) encodes a ProRS containing a truncated (non-editing) INS domain in addition to two single-domain homologs YbaK and ProX. Similar to Ec ProRS, Rp ProRS activates cognate Pro and misactivates noncognate Ala and Cys. Rp ProRS shows pre-transfer editing activity against Ala that is comparable to that of Ec ProRS and in the presence of tRNA, the synthetase robustly mischarges Ala onto tRNAPro. Therefore, despite the lack of a full INS domain and the relatively robust pre-transfer editing activity of Rp ProRS, we hypothesize that editing of Ala-tRNAPro is required. Additionally, efficient activation of Cys coupled with minimal Cys pre-transfer editing and good mischarging activity implies that editing of Cys-tRNAPro is essential. Here, we show that the mini-INS lacks hydrolytic activity, as expected, but appears to be critical for Rp ProRS structural stability. Also, Rp YbaK possesses robust Cys-tRNAPro editing activity. The function of ProX has not been reported for any species. We demonstrate here that Rp ProX inhibits mischarging of Ala on to tRNAPro in the presence of ProRS. Thus, in Rp an alternative triple sieve editing mechanism appears to exist in which the catalytic domain acts as the coarse sieve, and the single-domain proteins ProX and YbaK act as fine sieves to prevent formation of misacylated Ala-tRNAPro and Cys-tRNAPro, respectively.

Keywords: aminoacyl tRNA synthetase, post-transfer editing, tRNA