The Experts below are selected from a list of 297 Experts worldwide ranked by ideXlab platform
Peter G Schultz - One of the best experts on this subject based on the ideXlab platform.
-
an evolved Aminoacyl tRNA Synthetase with atypical polysubstrate specificity
Biochemistry, 2011Co-Authors: Douglas D Young, Glen Spraggon, Travis S Young, Michael Jahnz, Insha Ahmad, Peter G SchultzAbstract:We have employed a rapid fluorescence-based screen to assess the polyspecificity of several Aminoacyl-tRNA Synthetases (aaRSs) against an array of unnatural amino acids. We discovered that a p-cyanophenylalanine specific Aminoacyl-tRNA 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, 2008Co-Authors: Eric M Brustad, Mark L Bushey, Ansgar Brock, Johnathan Chittuluru, Peter G SchultzAbstract: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 Aminoacyl-tRNA 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, 2006Co-Authors: James M Turner, James Graziano, Glen Spraggon, Peter G SchultzAbstract:Recently, tRNA Aminoacyl-tRNA 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 Aminoacyl-tRNA 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 Aminoacyl-tRNA 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.
-
structural characterization of a p acetylphenylalanyl Aminoacyl tRNA Synthetase
Journal of the American Chemical Society, 2005Co-Authors: James M Turner, James Graziano, Glen Spraggon, Peter G SchultzAbstract:: It has been recently shown that orthogonal tRNA/Aminoacyl-tRNA Synthetase pairs can be evolved to allow genetic incorporation of unnatural amino acids into proteins in both prokaryotes and eukaryotes. Here we describe the crystal structure of an evolved Aminoacyl-tRNA Synthetase that charges the unnatural amino acid p-acetylphenylalanine. Molecular recognition is due to altered hydrogen bonding and packing interactions with bound substrate that result from changes in both side-chain and backbone conformation.
-
An efficient system for the evolution of Aminoacyl-tRNA Synthetase specificity.
Nature Biotechnology, 2002Co-Authors: Stephen W. Santoro, Lei Wang, Brad Herberich, David S. King, Peter G SchultzAbstract:A variety of strategies to incorporate unnatural amino acids into proteins have been pursued1,2,3,4,5, but all have limitations with respect to technical accessibility, scalability, applicability to in vivo studies, or site specificity of amino acid incorporation. The ability to selectively introduce unnatural functional groups into specific sites within proteins, in vivo6,7, provides a potentially powerful approach to the study of protein function and to large-scale production of novel proteins. Here we describe a combined genetic selection and screen that allows the rapid evolution of Aminoacyl-tRNA Synthetase substrate specificity. Our strategy involves the use of an “orthogonal” Aminoacyl-tRNA Synthetase and tRNA pair that cannot interact with any of the endogenous Synthetase–tRNA pairs in Escherichia coli8,9,10,11. A chloramphenicol-resistance (Cmr) reporter is used to select highly active Synthetase variants, and an amplifiable fluorescence reporter is used together with fluorescence-activated cell sorting (FACS) to screen for variants with the desired change in amino acid specificity. Both reporters are contained within a single genetic construct, eliminating the need for plasmid shuttling and allowing the evolution to be completed in a matter of days. Following evolution, the amplifiable fluorescence reporter allows visual and fluorimetric evaluation of Synthetase activity and selectivity. Using this system to explore the evolvability of an amino acid binding pocket of a tyrosyl-tRNA Synthetase, we identified three new variants that allow the selective incorporation of amino-, isopropyl-, and allyl-containing tyrosine analogs into a desired protein. The new enzymes can be used to produce milligram-per-liter quantities of unnatural amino acid–containing protein in E. coli.
Marc Mirande - One of the best experts on this subject based on the ideXlab platform.
-
The Aminoacyl-tRNA Synthetase Complex
Sub-cellular biochemistry, 2017Co-Authors: Marc MirandeAbstract:Aminoacyl-tRNA 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 Aminoacyl-tRNA 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-Aminoacyl-tRNA 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-Aminoacyl-tRNA 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 Aminoacyl-tRNA 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, 2016Co-Authors: Azaria Remion, Marc Mirande, Fawzi Khoderagha, David Cornu, Manuela Argentini, Virginie RedekerAbstract:Human cytoplasmic lysyl-tRNA Synthetase (LysRS) is associated within a multi-Aminoacyl-tRNA 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, 2015Co-Authors: Svitlana Havrylenko, Marc MirandeAbstract:Aminoacyl-tRNA 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-Aminoacyl-tRNA 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.
-
small angle x ray solution scattering study of the multi Aminoacyl tRNA Synthetase complex reveals an elongated and multi armed particle
Journal of Biological Chemistry, 2013Co-Authors: Jose Dias, Louis Renault, Javier Perez, Marc MirandeAbstract:Abstract In animal cells, nine Aminoacyl-tRNA Synthetases are associated with the three auxiliary proteins p18, p38, and p43 to form a stable and conserved large multi-Aminoacyl-tRNA Synthetase complex (MARS), whose molecular mass has been proposed to be between 1.0 and 1.5 MDa. The complex acts as a molecular hub for coordinating protein synthesis and diverse regulatory signal pathways. Electron microscopy studies defined its low resolution molecular envelope as an overall rather compact, asymmetric triangular shape. Here, we have analyzed the composition and homogeneity of the native mammalian MARS isolated from rabbit liver and characterized its overall internal structure, size, and shape at low resolution by hydrodynamic methods and small-angle x-ray scattering in solution. Our data reveal that the MARS exhibits a much more elongated and multi-armed shape than expected from previous reports. The hydrodynamic and structural features of the MARS are large compared with other supramolecular assemblies involved in translation, including ribosome. The large dimensions and non-compact structural organization of MARS favor a large protein surface accessibility for all its components. This may be essential to allow structural rearrangements between the catalytic and cis-acting tRNA binding domains of the Synthetases required for binding the bulky tRNA substrates. This non-compact architecture may also contribute to the spatiotemporal controlled release of some of its components, which participate in non-canonical functions after dissociation from the complex.
-
dynamic organization of Aminoacyl tRNA Synthetase complexes in the cytoplasm of human cells
Journal of Biological Chemistry, 2009Co-Authors: Monika Kaminska, Svitlana Havrylenko, Paulette Decottignies, Pierre Marechal, Boris Negrutskii, Marc MirandeAbstract:The localization in space and in time of proteins within the cytoplasm of eukaryotic cells is a central question of the cellular compartmentalization of metabolic pathways. The assembly of proteins within stable or transient complexes plays an essential role in this process. Here, we examined the subcellular localization of the multi-Aminoacyl-tRNA Synthetase complex in human cells. The sequestration of its components within the cytoplasm rests on the presence of the eukaryotic-specific polypeptide extensions that characterize the human enzymes, as compared with their prokaryotic counterparts. The cellular mobility of several Synthetases, assessed by measuring fluorescence recovery after photobleaching, suggested that they are not freely diffusible within the cytoplasm. Several of these enzymes, isolated by tandem affinity purification, were copurified with ribosomal proteins and actin. The capacity of Aminoacyl-tRNA Synthetases to interact with polyribosomes and with the actin cytoskeleton impacts their subcellular localization and mobility. Our observations have conceptual implications for understanding how translation machinery is organized in vivo.
Jason W Chin - One of the best experts on this subject based on the ideXlab platform.
-
rapid discovery and evolution of orthogonal Aminoacyl tRNA Synthetase tRNA pairs
Nature Biotechnology, 2020Co-Authors: Daniele Cervettini, Jason W Chin, Shan Tang, Stephen D Fried, Julian C W Willis, Louise Funke, Lucy J ColwellAbstract:A central challenge in expanding the genetic code of cells to incorporate noncanonical amino acids into proteins is the scalable discovery of Aminoacyl-tRNA 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 Aminoacyl-tRNA Synthetase/tRNA pairs.
Journal of the American Chemical Society, 2010Co-Authors: Heinz Neumann, Adrian L Slusarczyk, Jason W ChinAbstract:The genetic code sets the correspondence between codons and the amino acids they encode in protein translation. The code is enforced by Aminoacyl-tRNA 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 Aminoacyl-tRNA Synthetases and tRNAs from common ancestral Synthetase/tRNA pairs. This process produced the current set of mutually orthogonal Aminoacyl-tRNA 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, 2010Co-Authors: Heinz Neumann, Adrian L Slusarczyk, Jason W ChinAbstract:The genetic code sets the correspondence between codons and the amino acids they encode in protein translation. The code is enforced by Aminoacyl-tRNA 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 Aminoacyl-tRNA Synthetases and tRNAs from common ancestral Synthetase/tRNA pairs. This process produced the current set of mutually orthogonal Aminoacyl-tRNA 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...
Dieter Soll - One of the best experts on this subject based on the ideXlab platform.
-
Engineered Aminoacyl-tRNA Synthetases with Improved Selectivity toward Noncanonical Amino Acids.
ACS Chemical Biology, 2019Co-Authors: Hui Si Kwok, Oscar Vargas-rodriguez, Sergey V. Melnikov, Dieter SollAbstract:A wide range of noncanonical amino acids (ncAAs) can be incorporated into proteins in living cells by using engineered Aminoacyl-tRNA Synthetase/tRNA pairs. However, most engineered tRNA Synthetases are polyspecific; that is, they can recognize multiple rather than one ncAA. Polyspecificity of engineered tRNA Synthetases imposes a limit to the use of genetic code expansion because it prevents specific incorporation of a desired ncAA when multiple ncAAs are present in the growth media. In this study, we employed directed evolution to improve substrate selectivity of polyspecific tRNA Synthetases by developing substrate-selective readouts for flow-cytometry-based screening with the simultaneous presence of multiple ncAAs. We applied this method to improve the selectivity of two commonly used tRNA Synthetases, p-cyano-l-phenylalanyl Aminoacyl-tRNA Synthetase (pCNFRS) and Ne-acetyl-lysyl Aminoacyl-tRNA Synthetase (AcKRS), with broad specificity. Evolved pCNFRS and AcKRS variants exhibit significantly improved...
-
an Aminoacyl tRNA Synthetase that specifically activates pyrrolysine
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Carla Polycarpo, Alexandre Ambrogelly, Pamela F Crain, James A Mccloskey, Amelie Berube, Susann M Winbush, John L Wood, Dieter SollAbstract:Pyrrolysine, the 22nd cotranslationally inserted amino acid, was found in the Methanosarcina barkeri monomethylamine methyltransferase protein in a position that is encoded by an in-frame UAG stop codon in the mRNA. M. barkeri encodes a special amber suppressor tRNA (tRNAPyl) that presumably recognizes this UAG codon. It was reported that Lys-tRNAPyl can be formed by the Aminoacyl-tRNA Synthetase-like M. barkeri protein PylS [Srinivasan, G., James, C. M. & Krzycki, J. A. (2002) Science 296, 1459–1462], whereas a later article showed that Lys-tRNAPyl is synthesized by the combined action of LysRS1 and LysRS2, the two different M. barkeri lysyl-tRNA Synthetases. Pyrrolysyl-tRNAPyl formation was presumed to result from subsequent modification of lysine attached to tRNAPyl. To investigate whether pyrrolysine can be directly attached to tRNAPyl we chemically synthesized pyrrolysine. We show that PylS is a specialized Aminoacyl-tRNA Synthetase for charging pyrrolysine to tRNAPyl; lysine and tRNALys are not substrates of the enzyme. In view of the properties of PylS we propose to name this enzyme pyrrolysyl-tRNA Synthetase. In contrast, the LysRS1:LysRS2 complex does not recognize pyrrolysine and charges tRNAPyl with lysine. These in vitro data suggest that Methanosarcina cells have two pathways for acylating the suppressor tRNAPyl. This would ensure efficient translation of the in-frame UAG codon in case of pyrrolysine deficiency and safeguard the biosynthesis of the proteins whose genes contain this special codon.
-
a truncated Aminoacyl tRNA Synthetase modifies rna
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Juan C Salazar, Alexandre Ambrogelly, Pamela F Crain, James A Mccloskey, Dieter SollAbstract:Aminoacyl–tRNA Synthetases are modular enzymes composed of a central active site domain to which additional functional domains were appended in the course of evolution. Analysis of bacterial genome sequences revealed the presence of many shorter Aminoacyl–tRNA Synthetase paralogs. Here we report the characterization of a well conserved glutamyl–tRNA Synthetase (GluRS) paralog (YadB in Escherichia coli) that is present in the genomes of >40 species of proteobacteria, cyanobacteria, and actinobacteria. The E. coli yadB gene encodes a truncated GluRS that lacks the C-terminal third of the protein and, consequently, the anticodon binding domain. Generation of a yadB disruption showed the gene to be dispensable for E. coli growth in rich and minimal media. Unlike GluRS, the YadB protein was able to activate glutamate in presence of ATP in a tRNA-independent fashion and to transfer glutamate onto tRNAAsp. Neither tRNAGlu nor tRNAGln were substrates. In contrast to canonical Aminoacyl–tRNA, glutamate was not esterified to the 3′-terminal adenosine of tRNAAsp. Instead, it was attached to the 2-amino-5-(4,5-dihydroxy-2-cyclopenten-1-yl) moiety of queuosine, the modified nucleoside occupying the first anticodon position of tRNAAsp. Glutamyl–queuosine, like canonical Glu–tRNA, was hydrolyzed by mild alkaline treatment. Analysis of tRNA isolated under acidic conditions showed that this novel modification is present in normal E. coli tRNA; presumably it previously escaped detection as the standard conditions of tRNA isolation include an alkaline deacylation step that also causes hydrolysis of glutamyl–queuosine. Thus, this Aminoacyl–tRNA Synthetase fragment contributes to standard nucleotide modification of tRNA.
-
A truncated Aminoacyl–tRNA Synthetase modifies RNA
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Juan C Salazar, Alexandre Ambrogelly, Pamela F Crain, James A Mccloskey, Dieter SollAbstract:Aminoacyl–tRNA Synthetases are modular enzymes composed of a central active site domain to which additional functional domains were appended in the course of evolution. Analysis of bacterial genome sequences revealed the presence of many shorter Aminoacyl–tRNA Synthetase paralogs. Here we report the characterization of a well conserved glutamyl–tRNA Synthetase (GluRS) paralog (YadB in Escherichia coli) that is present in the genomes of >40 species of proteobacteria, cyanobacteria, and actinobacteria. The E. coli yadB gene encodes a truncated GluRS that lacks the C-terminal third of the protein and, consequently, the anticodon binding domain. Generation of a yadB disruption showed the gene to be dispensable for E. coli growth in rich and minimal media. Unlike GluRS, the YadB protein was able to activate glutamate in presence of ATP in a tRNA-independent fashion and to transfer glutamate onto tRNAAsp. Neither tRNAGlu nor tRNAGln were substrates. In contrast to canonical Aminoacyl–tRNA, glutamate was not esterified to the 3′-terminal adenosine of tRNAAsp. Instead, it was attached to the 2-amino-5-(4,5-dihydroxy-2-cyclopenten-1-yl) moiety of queuosine, the modified nucleoside occupying the first anticodon position of tRNAAsp. Glutamyl–queuosine, like canonical Glu–tRNA, was hydrolyzed by mild alkaline treatment. Analysis of tRNA isolated under acidic conditions showed that this novel modification is present in normal E. coli tRNA; presumably it previously escaped detection as the standard conditions of tRNA isolation include an alkaline deacylation step that also causes hydrolysis of glutamyl–queuosine. Thus, this Aminoacyl–tRNA Synthetase fragment contributes to standard nucleotide modification of tRNA.
-
a dual specificity Aminoacyl tRNA Synthetase in the deep rooted eukaryote giardia lamblia
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Shipra Bunjun, Constantinos Stathopoulos, David E Graham, Makoto Kitabatake, Alice L Wang, Ching C Wang, Christian P Vivares, Louis M Weiss, Dieter SollAbstract:Cysteinyl-tRNA (Cys-tRNA) is essential for protein synthesis. In most organisms the enzyme responsible for the formation of Cys-tRNA is cysteinyl-tRNA Synthetase (CysRS). The only known exceptions are the euryarchaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum, which do not encode a CysRS. Deviating from the accepted concept of one Aminoacyl-tRNA Synthetase per amino acid, these organisms employ prolyl-tRNA Synthetase as the enzyme that carries out Cys-tRNA formation. To date this dual-specificity prolyl-cysteinyl-tRNA Synthetase (ProCysRS) is only known to exist in archaea. Analysis of the preliminary genomic sequence of the primitive eukaryote Giardia lamblia indicated the presence of an archaeal prolyl-tRNA Synthetase (ProRS). Its proS gene was cloned and the gene product overexpressed in Escherichia coli. By using G. lamblia, M. jannaschii, or E. coli tRNA as substrate, this ProRS was able to form Cys-tRNA and Pro-tRNA in vitro. Cys-AMP formation, but not Pro-AMP synthesis, was tRNA-dependent. The in vitro data were confirmed in vivo, as the cloned G. lamblia proS gene was able to complement a temperature-sensitive E. coli cysS strain. Inhibition studies of CysRS activity with proline analogs (thiaproline and 5′-O-[N-(l-prolyl)-sulfamoyl]adenosine) in a Giardia S-100 extract predicted that the organism also contains a canonical CysRS. This prediction was confirmed by cloning and analysis of the corresponding cysS gene. Like a number of archaea, Giardia contains two enzymes, ProCysRS and CysRS, for Cys-tRNA formation. In contrast, the purified Saccharomyces cerevisiae and E. coli ProRS enzymes were unable to form Cys-tRNA under these conditions. Thus, the dual specificity is restricted to the archaeal genre of ProRS. G. lamblia's archaeal-type prolyl- and alanyl-tRNA Synthetases refine our understanding of the evolution and interaction of archaeal and eukaryal translation systems.
Kenneth Stuart - One of the best experts on this subject based on the ideXlab platform.
-
a multiple Aminoacyl tRNA Synthetase complex that enhances tRNA Aminoacylation in african trypanosomes
Molecular and Cellular Biology, 2013Co-Authors: Igor Cestari, Kenneth Stuart, Savitha Kalidas, Severine Monnerat, Atashi Anupama, Margaret A PhillipsAbstract:The genes for all cytoplasmic and potentially all mitochondrial Aminoacyl-tRNA Synthetases (aaRSs) were identified, and all those tested by RNA interference were found to be essential for the growth of Trypanosoma brucei. Some of these enzymes were localized to the cytoplasm or mitochondrion, but most were dually localized to both cellular compartments. Cytoplasmic T. brucei aaRSs were organized in a multiprotein complex in both bloodstream and procyclic forms. The multiple Aminoacyl-tRNA Synthetase (MARS) complex contained at least six aaRS enzymes and three additional non-aaRS proteins. Steady-state kinetic studies showed that association in the MARS complex enhances tRNA-Aminoacylation efficiency, which is in part dependent on a MARS complex-associated protein (MCP), named MCP2, that binds tRNAs and increases their Aminoacylation by the complex. Conditional repression of MCP2 in T. brucei bloodstream forms resulted in reduced parasite growth and infectivity in mice. Thus, association in a MARS complex enhances tRNA-Aminoacylation and contributes to parasite fitness. The MARS complex may be part of a cellular regulatory system and a target for drug development.
-
a spectrophotometric assay for quantitative measurement of Aminoacyl tRNA Synthetase activity
Journal of Biomolecular Screening, 2013Co-Authors: Igor Cestari, Kenneth StuartAbstract:Aminoacyl-tRNA Synthetases are enzymes that charge specific tRNAs with their cognate amino acids and play an essential role in the initial steps of protein synthesis. Because these enzymes are attractive targets for drug development in many microorganisms, there is a pressing need for assays suitable for compound screening. We developed (1) a high-throughput assay for measuring Aminoacyl-tRNA Synthetase activity and (2) an accompanying method for preparing the tRNA substrate. The assay can be performed in 96-well plates and relies on malachite green detection of pyrophosphate (Pi) as an indicator of Aminoacyl-tRNA Synthetase activity. Analysis of Trypanosoma brucei isoleucyl-tRNA Synthetase (IleRS) activity showed that the assay exhibits sensitivity to picomoles of product and yielded a Z′ factor of 0.56. We show that this assay is applicable to other Aminoacyl-tRNA Synthetases and to enzyme inhibition studies. Using this assay, we found that the compound NSC616354 inhibits recombinant IleRS with an IC50 ...
