Poster number 90 submitted by Anna Vradi
Discriminator Base is a Critical Recognition Element for Trypanosoma Brucei Ala-tRNAPro Editing Domains
Anna Vradi (Center for RNA Biology, Department of Microbiology, The Ohio State University Columbus, Ohio 43210), Rylan Watkins (Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University Columbus, Ohio 43210), Irina Shulgina (Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University Columbus, Ohio 43210), Karin Musier-Forsyth (Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University Columbus, Ohio 43210)
Aminoacyl-tRNA synthetases (ARSs) are responsible for charging tRNA with the proper amino acid. However, ARSs are error-prone and rely on editing mechanisms to lower the rate of misincorporation. Previous studies have shown prolyl-tRNA synthetase (ProRS) mischarges Ala onto cognate tRNAPro—this error can be corrected by a cis-editing site (INS) or by a freestanding INS homolog, ProXp-ala. Trypanosoma brucei (Tb) ProRS encodes an appended ProXp-ala domain, which is a putative cis-acting Ala-tRNAPro editing domain, as well as a distinct free-standing ProXp-ala homolog that we have named ProXp-ala2. Tb infection causes human African Trypanosomiasis (HAT), which is fatal; current treatments have serious side-effects and are relatively ineffective. ProXp-ala2 is not found in humans making it a potential drug target. Tb may have two Ala-tRNAPro editing domains due to its unusual metabolism. When in the procylic form, Tb’s main source of carbon is Pro, with Ala as a metabolic byproduct. With high concentrations of Ala, robust editing mechanisms may avoid misincorporation of Ala at Pro codons. Previous studies have shown that Homo Sapiens (Hs) ProXp-ala recognizes nucleotides in the tRNA acceptor stem for optimal function. In contrast, the E. coli INS domain, which is appended to the synthetase, relies exclusively on ProRS recognition of the anticodon. Our goal is to elucidate the acceptor stem specificity of Tb ProXp-ala, both free and when appended to ProRS, as well as of ProXp-ala2. We have mutated the acceptor stem of Tb tRNAPro to mimic a bacterial acceptor stem (G1C:C72G, C73A) and performed deacylation assays. Preliminary data suggest that both freestanding Tb ProXp-ala and ProXp-ala2 have strong specificity for the tRNAPro discriminator base (C73) and more relaxed specificity for the first base pair (G1:C72) when compared to similar experiments with Hs ProXp-ala. Surprisingly, ProXp-ala2 deacylated triple mutant (G1C:C72G, C73A) tRNA better than the single (C73A) mutant. Thus, the context of the discriminator base is important for this editing domain. Ongoing studies are aimed at elucidating the Tb ProXp-ala/2 residues responsible for acceptor stem binding. Results of these studies may have implications for new therapeutic strategies.
Keywords: tRNA Synthetase, tRNA, Trypanosoma Brucei