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Aminoacyl tRNA Synthetase

The Experts below are selected from a list of 297 Experts worldwide ranked by ideXlab platform

Peter G Schultz – 1st expert on this subject based on the ideXlab platform

  • an evolved Aminoacyl tRNA Synthetase with atypical polysubstrate specificity
    Biochemistry, 2011
    Co-Authors: Douglas D Young, Glen Spraggon, Travis S Young, Michael Jahnz, Insha Ahmad, Peter G Schultz

    Abstract:

    We have employed a rapid fluorescence-based screen to assess the polyspecificity of several AminoacyltRNA Synthetases (aaRSs) against an array of unnatural amino acids. We discovered that a p-cyanophenylalanine specific AminoacyltRNA Synthetase (pCNF-RS) has high substrate permissivity for unnatural amino acids, while maintaining its ability to discriminate against the 20 canonical amino acids. This orthogonal pCNF-RS, together with its cognate amber nonsense suppressor tRNA, is able to selectively incorporate 18 unnatural amino acids into proteins, including trifluoroketone-, alkynyl-, and halogen-substituted amino acids. In an attempt to improve our understanding of this polyspecificity, the X-ray crystal structure of the aaRS−p-cyanophenylalanine complex was determined. A comparison of this structure with those of other mutant aaRSs showed that both binding site size and other more subtle features control substrate polyspecificity.

  • a promiscuous Aminoacyl tRNA Synthetase that incorporates cysteine methionine and alanine homologs into proteins
    Bioorganic & Medicinal Chemistry Letters, 2008
    Co-Authors: Eric M Brustad, Mark L Bushey, Ansgar Brock, Johnathan Chittuluru, Peter G Schultz

    Abstract:

    Abstract A mutant Escherichia coli leucyl-tRNA Synthetase has been evolved for the selective incorporation of the methionine homolog 1 into proteins in yeast. This single AminoacyltRNA Synthetase is capable of charging an amber suppressor Ec tRNA CUA Leu with at least eight different amino acids including methionine and cysteine homologs, as well as straight chain aliphatic amino acids. In addition we show that incorporation yields for these amino acids can be increased substantially by mutations in the editing CP1 domain of the E. coli leucyl-tRNA Synthetase.

  • structural plasticity of an Aminoacyl tRNA Synthetase active site
    Proceedings of the National Academy of Sciences of the United States of America, 2006
    Co-Authors: James M Turner, James Graziano, Glen Spraggon, Peter G Schultz

    Abstract:

    Recently, tRNA AminoacyltRNA Synthetase pairs have been evolved that allow one to genetically encode a large array of unnatural amino acids in both prokaryotic and eukaryotic organisms. We have determined the crystal structures of two substrate-bound Methanococcus jannaschii tyrosyl AminoacyltRNA Synthetases that charge the unnatural amino acids p-bromophenylalanine and 3-(2-naphthyl)alanine (NpAla). A comparison of these structures with the substrate-bound WT Synthetase, as well as a mutant Synthetase that charges p-acetylphenylalanine, shows that altered specificity is due to both side-chain and backbone rearrangements within the active site that modify hydrogen bonds and packing interactions with substrate, as well as disrupt the α8-helix, which spans the WT active site. The high degree of structural plasticity that is observed in these AminoacyltRNA Synthetases is rarely found in other mutant enzymes with altered specificities and provides an explanation for the surprising adaptability of the genetic code to novel amino acids.

Marc Mirande – 2nd expert on this subject based on the ideXlab platform

  • The AminoacyltRNA Synthetase Complex
    Sub-cellular biochemistry, 2017
    Co-Authors: Marc Mirande

    Abstract:

    AminoacyltRNA Synthetases (AARSs) are essential enzymes that specifically Aminoacylate one tRNA molecule by the cognate amino acid. They are a family of twenty enzymes, one for each amino acid. By coupling an amino acid to a specific RNA triplet, the anticodon, they are responsible for interpretation of the genetic code. In addition to this translational, canonical role, several AminoacyltRNA Synthetases also fulfill nontranslational, moonlighting functions. In mammals, nine Synthetases, those specific for amino acids Arg, Asp, Gln, Glu, Ile, Leu, Lys, Met and Pro, associate into a multi-AminoacyltRNA Synthetase complex, an association which is believed to play a key role in the cellular organization of translation, but also in the regulation of the translational and nontranslational functions of these enzymes. Because the balance between their alternative functions rests on the assembly and disassembly of this supramolecular entity, it is essential to get precise insight into the structural organization of this complex. The high-resolution 3D-structure of the native particle, with a molecular weight of about 1.5 MDa, is not yet known. Low-resolution structures of the multi-AminoacyltRNA Synthetase complex, as determined by cryo-EM or SAXS, have been reported. High-resolution data have been reported for individual enzymes of the complex, or for small subcomplexes. This review aims to present a critical view of our present knowledge of the AminoacyltRNA Synthetase complex in 3D. These preliminary data shed some light on the mechanisms responsible for the balance between the translational and nontranslational functions of some of its components.

  • identification of protein interfaces within the multi Aminoacyl tRNA Synthetase complex the case of lysyl tRNA Synthetase and the scaffold protein p38
    FEBS Open Bio, 2016
    Co-Authors: Azaria Remion, Marc Mirande, Fawzi Khoderagha, David Cornu, Manuela Argentini, Virginie Redeker

    Abstract:

    Human cytoplasmic lysyl-tRNA Synthetase (LysRS) is associated within a multi-AminoacyltRNA Synthetase complex (MSC). Within this complex, the p38 component is the scaffold protein that binds the catalytic domain of LysRS via its N-terminal region. In addition to its translational function when associated to the MSC, LysRS is also recruited in nontranslational roles after dissociation from the MSC. The balance between its MSC-associated and MSC-dissociated states is essential to regulate the functions of LysRS in cellular homeostasis. With the aim of understanding the rules that govern association of LysRS in the MSC, we analyzed the protein interfaces between LysRS and the full-length version of p38, the scaffold protein of the MSC. In a previous study, the cocrystal structure of LysRS with a N-terminal peptide of p38 was reported [Ofir-Birin Y et al. (2013) Mol Cell 49, 30-42]. In order to identify amino acid residues involved in interaction of the two proteins, the non-natural, photo-cross-linkable amino acid p-benzoyl-l-phenylalanine (Bpa) was incorporated at 27 discrete positions within the catalytic domain of LysRS. Among the 27 distinct LysRS mutants, only those with Bpa inserted in place of Lys356 or His364 were cross-linked with p38. Using mass spectrometry, we unambiguously identified the protein interface of the cross-linked complex and showed that Lys356 and His364 of LysRS interact with the peptide from Pro8 to Arg26 in native p38, in agreement with the published cocrystal structure. This interface, which in LysRS is located on the opposite side of the dimer to the site of interaction with its tRNA substrate, defines the core region of the MSC. The residues identified herein in human LysRS are not conserved in yeast LysRS, an enzyme that does not associate within the MSC, and contrast with the residues proposed to be essential for LysRS:p38 association in the earlier work.

  • Aminoacyl tRNA Synthetase complexes in evolution
    International Journal of Molecular Sciences, 2015
    Co-Authors: Svitlana Havrylenko, Marc Mirande

    Abstract:

    AminoacyltRNA Synthetases are essential enzymes for interpreting the genetic code. They are responsible for the proper pairing of codons on mRNA with amino acids. In addition to this canonical, translational function, they are also involved in the control of many cellular pathways essential for the maintenance of cellular homeostasis. Association of several of these enzymes within supramolecular assemblies is a key feature of organization of the translation apparatus in eukaryotes. It could be a means to control their oscillation between translational functions, when associated within a multi-AminoacyltRNA Synthetase complex (MARS), and nontranslational functions, after dissociation from the MARS and association with other partners. In this review, we summarize the composition of the different MARS described from archaea to mammals, the mode of assembly of these complexes, and their roles in maintenance of cellular homeostasis.

Jason W Chin – 3rd expert on this subject based on the ideXlab platform

  • rapid discovery and evolution of orthogonal Aminoacyl tRNA Synthetase tRNA pairs
    Nature Biotechnology, 2020
    Co-Authors: Daniele Cervettini, Jason W Chin, Shan Tang, Stephen D Fried, Julian C W Willis, Louise Funke, Lucy J Colwell

    Abstract:

    A central challenge in expanding the genetic code of cells to incorporate noncanonical amino acids into proteins is the scalable discovery of AminoacyltRNA Synthetase (aaRS)-tRNA pairs that are orthogonal in their Aminoacylation specificity. Here we computationally identify candidate orthogonal tRNAs from millions of sequences and develop a rapid, scalable approach-named tRNA Extension (tREX)-to determine the in vivo Aminoacylation status of tRNAs. Using tREX, we test 243 candidate tRNAs in Escherichia coli and identify 71 orthogonal tRNAs, covering 16 isoacceptor classes, and 23 functional orthogonal tRNA-cognate aaRS pairs. We discover five orthogonal pairs, including three highly active amber suppressors, and evolve new amino acid substrate specificities for two pairs. Finally, we use tREX to characterize a matrix of 64 orthogonal Synthetase-orthogonal tRNA specificities. This work expands the number of orthogonal pairs available for genetic code expansion and provides a pipeline for the discovery of additional orthogonal pairs and a foundation for encoding the cellular synthesis of noncanonical biopolymers.

  • De novo generation of mutually orthogonal AminoacyltRNA Synthetase/tRNA pairs.
    Journal of the American Chemical Society, 2010
    Co-Authors: Heinz Neumann, Adrian L Slusarczyk, Jason W Chin

    Abstract:

    The genetic code sets the correspondence between codons and the amino acids they encode in protein translation. The code is enforced by AminoacyltRNA Synthetase/tRNA pairs, which direct the unique coupling of specific amino acids with specific anticodons. The evolutionary record suggests that a primitive genetic code expanded into the current genetic code, over billions of years, through duplication and specialization (neofunctionalization) of AminoacyltRNA Synthetases and tRNAs from common ancestral Synthetase/tRNA pairs. This process produced the current set of mutually orthogonal AminoacyltRNA Synthetases and tRNAs that direct natural protein synthesis. Here we demonstrate the creation of new orthogonal pairs, which are mutually orthogonal with existing orthogonal pairs, de novo, by a logical series of steps implemented in the laboratory, via the de novo generation of orthogonality in RNA−RNA interactions, protein−RNA interactions, and small molecule substrate selection by protein catalysts. Our lab…

  • de novo generation of mutually orthogonal Aminoacyl tRNA Synthetase tRNA pairs
    Journal of the American Chemical Society, 2010
    Co-Authors: Heinz Neumann, Adrian L Slusarczyk, Jason W Chin

    Abstract:

    The genetic code sets the correspondence between codons and the amino acids they encode in protein translation. The code is enforced by AminoacyltRNA Synthetase/tRNA pairs, which direct the unique coupling of specific amino acids with specific anticodons. The evolutionary record suggests that a primitive genetic code expanded into the current genetic code, over billions of years, through duplication and specialization (neofunctionalization) of AminoacyltRNA Synthetases and tRNAs from common ancestral Synthetase/tRNA pairs. This process produced the current set of mutually orthogonal AminoacyltRNA Synthetases and tRNAs that direct natural protein synthesis. Here we demonstrate the creation of new orthogonal pairs, which are mutually orthogonal with existing orthogonal pairs, de novo, by a logical series of steps implemented in the laboratory, via the de novo generation of orthogonality in RNA−RNA interactions, protein−RNA interactions, and small molecule substrate selection by protein catalysts. Our lab…