scholarly journals The isolation of a peptide from the catalytic domain of Bacillus stearothermophilus tryptophyl-tRNA synthetase. The interaction of Brown MX-5BR with tyrosyl-tRNA synthetase

1987 ◽  
Vol 243 (3) ◽  
pp. 701-707 ◽  
Author(s):  
J E C McArdell ◽  
C J Bruton ◽  
T Atkinson
Keyword(s):  
Amino Acid ◽  
Bacillus Stearothermophilus ◽  
Catalytic Domain ◽  
Competitive Inhibitor ◽  
Trna Synthetase ◽  
Apparent Dissociation Constant ◽  
Peptide Peak ◽  
Competitive Kinetics ◽  
Amino Acid Binding ◽  
Dissociation Constant Kd

Tryptophyl-tRNA synthetase is irreversibly inactivated by Procion Brown MX-5BR with an apparent dissociation constant (KD) of 8.8 microM and maximum rate of inactivation k3 0.192 s-1. The specificity of the interaction is supported by two previously reported observations. Firstly, Brown MX-5BR inactivation of tryptophyl-tRNA synthetase is inhibited by substrates, and secondly, the animated derivative of Brown MX-5BR is a competitive inhibitor of tryptophyl-tRNA synthetase with a Ki of 2 X 10(-4) M with respect to both tryptophan and ATP. Tryptic digestion of the dye-affinity-labelled enzyme and subsequent resolution of the peptides by h.p.l.c. yielded one major dye-peptide peak. Amino acid sequence analysis resulted in the identification of the dye-binding domain centred on lysine-178. Tyrosyl-tRNA synthetase is also inactivated by Procion Brown MX-5BR, and this inactivation is prevented by ATP but not by tyrosine. The interaction of tyrosyl-tRNA synthetase with hydroxylated Brown MX-5BR exhibited non-competitive kinetics with respect to the amino acid-binding site and competitive kinetics against ATP with a Ki of 6 X 10(-6) M.

scholarly journals Transcription and translation in an RNA world

2006 ◽  
Vol 361 (1474) ◽  
pp. 1751-1760 ◽  
Author(s):  
William R Taylor
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Rna World ◽  
Large Subunit ◽  
Trna Synthetase ◽  
Nucleoside Triphosphate ◽  
Amino Acid Binding ◽  
World Hypothesis ◽  
Protein Elongation ◽  
Rna World Hypothesis

The RNA world hypothesis requires a ribozyme that was an RNA-directed RNA polymerase (ribopolymerase). If such a replicase makes a reverse complementary copy of any sequence (including itself), in a simple RNA world, there is no mechanism to prevent self-hybridization. It is proposed that this can be avoided through the synthesis of a parallel complementary copy. The logical consequences of this are pursued and developed in a computer simulation, where the behaviour of the parallel copy is compared to the conventional reverse complementary copy. It is found that the parallel copy is more efficient at higher temperatures (up to 90°C). A model for the ribopolymerase, based on the core of the large subunit (LSU) of the ribosome, is described. The geometry of a potential active site for this ribopolymerase suggests that it contained a cavity (now occupied by the aminoacyl-tRNA) and that an amino acid binding in this might have ‘poisoned’ the ribopolymerase by cross-reacting with the nucleoside-triphosphate before polymerization could occur. Based on a similarity to the active site components of the class-I tRNA synthetase enzymes, it is proposed that the amino acid could become attached to the nascent RNA transcript producing a variety of aminoacylated tRNA-like products. Using base-pairing interactions, some of these molecules might cross-link two ribopolymerases, giving rise to a precursor of the modern ribosome. A hybrid dimer, half polymerase and half proto-ribosome, could account for mRNA translocation before the advent of protein elongation factors.

scholarly journals Pyrrolysyl-tRNA Synthetase with a Unique Architecture Enhances the Availability of Lysine Derivatives in Synthetic Genetic Codes

Molecules ◽  
2018 ◽  
Vol 23 (10) ◽  
pp. 2460 ◽  
Author(s):  
Atsushi Yamaguchi ◽  
Fumie Iraha ◽  
Kazumasa Ohtake ◽  
Kensaku Sakamoto
Keyword(s):  
Amino Acid ◽  
Binding Pocket ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
Genetic Codes ◽  
Genetic Code Expansion ◽  
Amino Acid Binding ◽  
Derivatives Of ◽  
Additional Nucleotide ◽  
Incorporation Efficiency

Genetic code expansion has largely relied on two types of the tRNA—aminoacyl-tRNA synthetase pairs. One involves pyrrolysyl-tRNA synthetase (PylRS), which is used to incorporate various lysine derivatives into proteins. The widely used PylRS from Methanosarcinaceae comprises two distinct domains while the bacterial molecules consist of two separate polypeptides. The recently identified PylRS from Candidatus Methanomethylophilus alvus (CMaPylRS) is a single-domain, one-polypeptide enzyme that belongs to a third category. In the present study, we showed that the PylRS—tRNAPyl pair from C. M. alvus can incorporate lysine derivatives much more efficiently (up to 14-times) than Methanosarcinaceae PylRSs in Escherichia coli cell-based and cell-free systems. Then we investigated the tRNA and amino-acid recognition by CMaPylRS. The cognate tRNAPyl has two structural idiosyncrasies: no connecting nucleotide between the acceptor and D stems and an additional nucleotide in the anticodon stem and it was found that these features are hardly recognized by CMaPylRS. Lastly, the Tyr126Ala and Met129Leu substitutions at the amino-acid binding pocket were shown to allow CMaPylRS to recognize various derivatives of the bulky Nε-benzyloxycarbonyl-l-lysine (ZLys). With the high incorporation efficiency and the amenability to engineering, CMaPylRS would enhance the availability of lysine derivatives in expanded codes.

scholarly journals Probing the substrate-binding sites of aminoacyl-tRNA synthetases with the procion dye green HE-4BD

1989 ◽  
Vol 258 (3) ◽  
pp. 715-721 ◽  
Author(s):  
J E C McArdell ◽  
M Duffield ◽  
T Atkinson
Keyword(s):  
Bacillus Stearothermophilus ◽  
Competitive Inhibitor ◽  
Reactive Dye ◽  
Trna Synthetase ◽  
Enzyme Inactivation ◽  
Trna Synthetases ◽  
Time Period ◽  
Substrate Binding Sites ◽  
Methionyl Trna Synthetase ◽  
Fold Excess

A reactive bis-dichloro derivative of the Procion dye Green HE-4BD was shown to inactivate irreversibly methionyl-tRNA synthetase (MTS) from Escherichia coli and also tryptophyl-tRNA synthetase (WTS) and tyrosyl-tRNA synthetase (YTS) from Bacillus stearothermophilus at pH 8.5 and 37 degrees C. At a 5-fold excess of reactive dye over enzyme subunit concentration MTS was quantitatively inactivated within 20 min in the ATP/pyrophosphate exchange assay, whereas WTS and YTS show an 80% loss of activity over the same time period. The inactivation is affected by the addition of substrates, which either protect (WTS and YTS) or promote (YTS with tyrosine) the dye-mediated enzyme inactivation. Green HE-4BD-OH was shown to be a competitive inhibitor of MTS with respect to MgATP, methionine and tRNA substrates.

Escherichia coliTryptophanyl-tRNA Synthetase Mutants Selected for Tryptophan Auxotrophy Implicate the Dimer Interface in Optimizing Amino Acid Binding†

Biochemistry ◽  
1996 ◽  
Vol 35 (1) ◽  
pp. 32-40 ◽  
Author(s):  
Sanja Sever ◽  
Kelley Rogers ◽  
M. John Rogers ◽  
Charles Carter, ◽  
Dieter Söll
Keyword(s):  
Amino Acid ◽  
Trna Synthetase ◽  
Dimer Interface ◽  
Amino Acid Binding

scholarly journals Arginyl-tRNA synthetase with signature sequence KMSK from Bacillus stearothermophilus

2003 ◽  
Vol 376 (3) ◽  
pp. 773-779 ◽  
Author(s):  
Juan LI ◽  
Yong-Neng YAO ◽  
Mo-Fang LIU ◽  
En-Duo WANG
Keyword(s):  
Amino Acid ◽  
Bacillus Stearothermophilus ◽  
Genomic Sequence ◽  
Trna Synthetase ◽  
Class I ◽  
Acid Activation ◽  
Gene Encoding ◽  
Mutual Compensation ◽  
Nickel Affinity Chromatography ◽  
High Loop

ArgRS (arginyl-tRNA synthetase) belongs to the class I aaRSs (aminoacyl-tRNA synthetases), though the majority of ArgRS species lack the canonical KMSK sequence characteristic of class I aaRSs. A DNA fragment of the ArgRS gene from Bacillus stearothermophilus was amplified using primers designed according to the conserved regions of known ArgRSs. Through analysis of the amplified DNA sequence and known tRNAArgs with a published genomic sequence of B. stearothermophilus, the gene encoding ArgRS (argS´) was amplified by PCR and the gene encoding tRNAArg (ACG) was synthesized. ArgRS contained 557 amino acid residues including the canonical KMKS sequence. Recombinant ArgRS and tRNAArg (ACG) were expressed in Escherichia coli. ArgRS purified by nickel-affinity chromatography had no ATPase activity. The kinetics of ArgRS and cross-recognition between ArgRSs and tRNAArgs from B. stearothermophilus and E. coli were studied. The activities of B. stearothermophilus ArgRS mutated at Lys382 and Lys385 of the KMSK sequence and at Gly136 upstream of the HIGH loop were determined. From the mutation results, we concluded that there was mutual compensation of Lys385 and Gly136 for the amino acid-activation activity of B. stearothermophilus ArgRS.

scholarly journals Isolation of the Gene Encoding the Drosophila melanogaster Homolog of the Saccharomyces cerevisiae GCN2 eIF-2α Kinase

Genetics ◽  
1998 ◽  
Vol 149 (3) ◽  
pp. 1495-1509 ◽  
Author(s):  
DeAnne S Olsen ◽  
Barbara Jordan ◽  
Dreeny Chen ◽  
Ronald C Wek ◽  
Douglas R Cavener
Keyword(s):  
Drosophila Melanogaster ◽  
Amino Acid ◽  
Catalytic Domain ◽  
Phosphorylation Site ◽  
Trna Synthetase ◽  
Cdna Clones ◽  
Gene Encoding ◽  
Partial Conservation ◽  
Higher Eukaryotes ◽  
The Right

Abstract Genomic and cDNA clones homologous to the yeast GCN2 eIF-2α kinase (yGCN2) were isolated from Drosophila melanogaster. The identity of the Drosophila GCN2 (dGCN2) gene is supported by the unique combination of sequence encoding a protein kinase catalytic domain and a domain homologous to histidyl-tRNA synthetase and by the ability of dGCN2 to complement a deletion mutant of the yeast GCN2 gene. Complementation of Δgcn2 in yeast by dGCN2 depends on the presence of the critical regulatory phosphorylation site (serine 51) of eIF-2α. dGCN2 is composed of 10 exons encoding a protein of 1589 amino acids. dGCN2 mRNA is expressed throughout Drosophila development and is particularly abundant at the earliest stages of embryogenesis. The dGCN2 gene was cytogenetically and physically mapped to the right arm of the third chromosome at 100C3 in STS Dm2514. The discovery of GCN2 in higher eukaryotes is somewhat unexpected given the marked differences between the amino acid biosynthetic pathways of yeast vs. Drosophila and other higher eukaryotes. Despite these differences, the presence of GCN2 in Drosophila suggests at least partial conservation from yeast to multicellular organisms of the mechanisms responding to amino acid deprivation.

The zinc-binding site of a class I aminoacyl-tRNA synthetase is a SWIM domain that modulates amino acid binding via the tRNA acceptor arm

2004 ◽  
Vol 271 (4) ◽  
pp. 724-733 ◽  
Author(s):  
Rajat Banerjee ◽  
Daniel Y. Dubois ◽  
Joelle Gauthier ◽  
Sheng-Xiang Lin ◽  
Siddhartha Roy ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Trna Synthetase ◽  
Class I ◽  
Zinc Binding ◽  
Aminoacyl Trna Synthetase ◽  
Zinc Binding Site ◽  
Amino Acid Binding ◽  
Trna Acceptor
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