Biology subjects

Vargas-Rodriguez, O.

Publications and source records attributed to Vargas-Rodriguez, O..

4 recordsLinked to original sources

bioRxiv2025

Mechanistic and evolutionary insights into a family of aminoacyl-tRNA deacylases that protects against canavanine toxicity

Aminoacyl-tRNA deacylases safeguard the accurate translation of the genetic code by hydrolyzing incorrectly synthesized aminoacyl-tRNAs. Canavanyl-tRNA deacylase (CtdA) was recently shown to protect cells against the toxicity of the non-proteinogenic amino acid canavanine, which is synthesized and accumulated by various plants. In most organisms, canavanine is ligated to tRNAArg, causing translation of arginine codons with canavanine. CtdA prevents canavanine toxicity by hydrolyzing canavanyl-tRNAArg. Here, we investigated the function, structure, substrate specificity, phylogenetic distribution, and evolution of CtdA. We show that CtdA is essential to prevent canavanine cytotoxicity in Salmonella enterica, and its heterologous expression can also rescue Escherichia coli. By determining the structure of CtdA, we identified its putative binding pocket and residues that modulate enzymatic activity and specificity. We also found that CtdA displays a relaxed specificity for the aminoacyl moiety substrate as it hydrolyzes arginyl-tRNAArg. Finally, we showed that despite their structural homology, CtdA and the aminoacyl-tRNA hydrolytic domain of phenylalanyl-tRNA synthetase are functionally and evolutionarily divergent. Collectively, these results substantially expand our understanding of the CtdA family, providing new insights into its structure, function, and evolution. Moreover, this work highlights the diverse mechanisms unique to each organism to ensure faithful translation of the genetic code.

biochemistry↗

bioRxiv2020

Genetic Encoding of Three Distinct Noncanonical Amino Acids Using Reprogrammed Initiator and Nonsense Codons

We recently described an orthogonal initiator tRNA (itRNATy2) that can initiate protein synthesis with noncanonical amino acids (ncAAs) in response to the UAG nonsense codon. Here we report that a mutant of itRNATy2 (itRNATy2AUA) can efficiently initiate translation in response to the UAU tyrosine codon, giving rise to proteins with an ncAA at their N-terminus. We show that, in cells expressing itRNATy2AUA, UAU can function as a dual-use codon that selectively encodes ncAAs at the initiating position and tyrosine at elongating positions. Using itRNATy2AUA, in conjunction with its cognate tyrosyl-tRNA synthetase and two mutually orthogonal pyrrolysyl-tRNA synthetases, we demonstrate that UAU can be reassigned along with UAG or UAA to encode two distinct ncAAs in the same protein. Furthermore, by engineering the substrate specificity of one of the pyrrolysyl-tRNA synthetases, we developed a triply orthogonal system that enables simultaneous reassignment of UAU, UAG, and UAA to produce proteins containing three distinct ncAAs at precisely defined sites. To showcase the utility of this system, we produced proteins containing two or three ncAAs, with unique bioorthogonal functional groups, and demonstrate that these proteins can be separately modified with multiple fluorescent probes. TOC Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/415521v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@10e7ebdorg.highwire.dtl.DTLVardef@a2c26dorg.highwire.dtl.DTLVardef@18ab3aborg.highwire.dtl.DTLVardef@119cf87_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2020

Human trans-editing enzyme displays tRNA acceptor stem specificity and relaxed amino acid selectivity

Accurate translation of genetic information into proteins is vital for cell sustainability. ProXp-ala prevents proteome-wide Pro-to-Ala mutations by hydrolyzing misacylated Ala-tRNAPro, which is synthesized by prolyl-tRNA synthetase (ProRS). Bacterial ProXp-ala was previously shown to combine a size-based exclusion mechanism with conformational and chemical selection for the recognition of the alanyl moiety, while tRNAPro is selected via recognition of tRNA acceptor stem elements G72 and A73. The identity of these critical bases changed during evolution with eukaryotic cytosolic tRNAPro possessing a cytosine at the corresponding positions. The mechanism by which eukaryotic ProXp-ala adapted to these changes remains unknown. In this work, recognition of the aminoacyl moiety and tRNA acceptor stem by human (Hs) ProXp-ala was examined. Enzymatic assays revealed that Hs ProXp-ala requires C72 and C73 in the context of Hs cytosolic tRNAPro for efficient deacylation of mischarged Ala-tRNAPro. The strong dependence on these bases prevents cross-species deacylation of bacterial Ala-tRNAPro or of Hs mitochondrial Ala-tRNAPro by the human enzyme. Similar to the bacterial enzyme, Hs ProXp-ala showed strong tRNA acceptor-stem recognition but differed in its amino acid specificity profile relative to bacterial ProXp-ala. Changes at conserved residues in both the Hs and bacterial ProXp-ala substrate binding pockets modulated this specificity. These results illustrate how the mechanism of substrate selection diverged during the evolution of the ProXp-ala family and provides the first example of a trans-editing domain whose specificity evolved to adapt to changes in its tRNA substrate.

biochemistry↗

bioRxiv2017

Engineering post-translational proofreading to discriminate non-standard amino acids

Progress in genetic code expansion requires accurate, selective, and high-throughput detection of non-standard amino acid (NSAA) incorporation into proteins. Here, we discover how the N-end rule pathway of protein degradation applies to commonly used NSAAs. We show that several NSAAs are N-end stabilizing and demonstrate that other NSAAs can be made stabilizing by rationally engineering the N-end rule adaptor protein ClpS. We use these insights to engineer a synthetic quality control method, termed \"Post-Translational Proofreading\" (PTP). By implementing PTP, false positive proteins resulting from misincorporation of structurally similar standard amino acids or undesired NSAAs rapidly degrade, enabling high-accuracy discrimination of desired NSAA incorporation. We illustrate the utility of PTP during evolution of the biphenylalanine orthogonal translation system used for synthetic biocontainment. Our new OTS is more selective and confers lower escape frequencies and greater fitness in all tested biocontained strains. Our approach presents a new paradigm for molecular recognition of amino acids in target proteins.

synthetic biology↗