Date of Award
Master of Science (MS)
Department of Chemistry
Christopher S Weitzel
Aminoacyl-transfer RNA synthetases (aaRSs) catalyze the attachment of an amino acid to the 3’-end of a cognate tRNA in a two-step reaction, fulfilling a pivotal role in RNA translation by providing “charged” tRNA for protein synthesis. Synthetases possess a catalytic core where amino acids are activated via adenylation with the subsequent transfer of that activated amino acid to the acceptor adenosine at position 76 of the tRNA sequence.1,2 The fidelity of tRNA aminoacylation is essential for faithful protein synthesis and requires the aaRS to discriminate against chemically and structurally similar amino acids, though some studies suggest that aaRS-dependent errors can be beneficial to some microbial species.3The meticulous accuracy of aaRS is dependent in part upon amino acid editing mechanisms. The aliphatic synthetases rely on hydrolytic editing activity to clear mischarged tRNA (post-transfer), performed in the connective polypeptide 1 (CP1) domain, distinct from the synthetic aminoacylation site. A second editing pathway hydrolyzes the aminoacyl adenylate intermediate at the synthetic site before transfer of the amino acid to the tRNA (pre-transfer).4 Both pre- and post-transfer editing capacity can co-exist in a single aaRS, though one is generally dominant in standard conditions. However, when an editing pathway is unavailable, the secondary pathway may be stimulated to suppress aaRS infidelity.5Bioinformatic analyses identified two distinct leucyl-tRNA synthetase genes within all genomes of the Archaeal family Sulfolobaceae.6 One copy, named LeuRS-I, has substitutions of key amino acids within its CP1 editing domain that would be expected to eliminate its ability to post-transfer edit mischarged tRNALeu, which could bring about variation within the proteome of these extremophiles. The other copy, LeuRS-F, contains canonical active sites in both the catalytic core and CP1 domain.6 Biochemical analysis of the paralogs within Sulfolobus islandicus supports the hypothesis that LeuRS-F, and not LeuRS-I, functions as an essential tRNA synthetase that faithfully charges leucine to tRNALeu. Remarkably, the ubiquitous conservation of LeuRS-I across the Sulfolobaceae, despite its inability to perform canonical synthetase activity, indicates an alternative, non-canonical function of this paralog. Further analysis of the charging and hydrolytic editing capability of these paralogs was performed by selective mutation of key residues in the CP1 domain of LeuRS-F to match those in LeuRS-I. Interestingly, elimination of the post-transfer editing capability of LeuRS-F does not result in mischarged tRNA, suggesting stimulation of a pre-transfer editing mechanism not commonly observed in LeuRS. Additional enzymatic analysis shows activation of non-cognate amino acids by both LeuRSs, a secondary tRNA dependent activity to prevent mischarging, and a defunct post-transfer editing capability for the LeuRS-F CP1-domain mutant.
Strandquist, Evan T., "Characterizing the Editing Modes of Duplicated Leucyl-Trna Synthetases in the Archaea Sulfolobus Islandicus" (2023). Theses and Dissertations. 1709.