Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.5.1.3 (dihydrofolate reductase)
5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Yeast tRNA ligase possesses multiple activities which are required for the joining of tRNA halves during the tRNA splicing process: cyclic phosphodiesterase, kinase, adenylylate synthetase, and ligase. A deletion polypeptide of a dihydrofolate reductase-ligase fusion protein, designated DAC, was previously shown to join tRNA halves although ATP-dependent kinase activity was not measurable in the assay used. We describe here a characterization of the mechanism of joining used by DAC and the structure of the tRNA product. DAC produces a joined tRNA and a splice junction with a structure identical to that produced by DAKC, the full-length dihydrofolate reductase-ligase fusion. Furthermore, DAC can use GTP as the sole cofactor in the joining reaction, in contrast to DAKC, which can only complete splicing in the presence of ATP. Both enzymes exhibit GTP-dependent kinase activity at 100-fold greater efficiency than with ATP. These results suggest that a potential function for the center domain of tRNA ligase (missing in DAC) is to provide structural integrity and aid in substrate interactions and specificity. They also support the hypothesis that ligase may prefer to use two different cofactors during tRNA splicing.
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PMID:Novel activity of a yeast ligase deletion polypeptide. Evidence for GTP-dependent tRNA splicing. 842 18

We have examined multiple cofactor usage by yeast tRNA ligase in splicing in vitro. The ligase mechanism of action requires expenditure of two molar equivalents of nucleotide cofactor per mole of tRNA product. Recent evidence (Westaway, S.K., Belford, H.G., Apostol, B.L., Abelson, J., and Greer, C.L. (1993) J. Biol. Chem. 268, 2435-2443) demonstrated that the ligase-associated kinase activity is more efficient with GTP as cofactor than with ATP. Employing a ligase fusion construct with dihydrofolate reductase (Apostol, B.L., Westaway, S.K., Abelson, J., and Greer, C.L. (1991) J. Biol. Chem. 266, 7445-7455) for purposes of enzyme purification, we performed joining assays demonstrating that ATP and GTP are the most effective combination of cofactors. ATP was essential to the joining reaction, while UTP, CTP, or ATP replaced GTP inefficiently. Specific and functionally independent binding sites were confirmed for ATP and GTP by direct binding measurement. A third site was implicated in UTP- and CTP-ligase interactions. Comparison of binding constants with Kapp values determined for nucleotide-dependent joining suggested both that nucleotide triphosphate binding may be limiting in tRNA joining and that tRNA ligation occurs most efficiently using GTP for the kinase reaction and ATP as the adenylylate synthetase cofactor.
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PMID:Multiple nucleotide cofactor use by yeast ligase in tRNA splicing. Evidence for independent ATP- and GTP-binding sites. 842 19

The nucleotide sequence of a 37 000 base pair region from the left arm of chromosome XV of Saccharomyces cerevisiae has been determined and analysed. This region contains 21 open reading frames (ORFs) coding for proteins of more than 100 amino acids. Six ORFs correspond to the genes PAC1, VPH1, MOD5, CAP20, ORF1 and SNF2 already described. Eight ORFs show some similarities to known genes from yeast and other organisms. They include genes coding for serine/threonine protein kinases, a multidrug resistance family homologue, a protein related to dihydrofolate reductase, a cluster of heat shock-like proteins and a gene coding for an enzyme related to protein disulfide isomerase. Finally seven ORFs do not show any similarities with a known gene. In addition we found a new ala-tRNA (UGC) gene located next to a sigma sequence.
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PMID:DNA sequence analysis of the VPH1-SNF2 region on chromosome XV of Saccharomyces cerevisiae. 889 71

Site-directed incorporation of the amino acid analogue p-fluoro-phenylalanine (p-F-Phe) was achieved in Escherichia coli. A yeast suppressor tRNA(Phe)amber/phenylalanyl-tRNA synthetase pair was expressed in an analogue-resistant E. coli strain to direct analogue incorporation at a programmed amber stop codon in the DHFR marker protein. The programmed position was translated to 64-75% as p-F-Phe and the remainder as phenylalanine and lysine. Depending on the expression conditions, the p-F-Phe incorporation was 11-21-fold higher at the programmed position than the background incorporation at phenylalanine codons, showing high specificity of analogue incorporation. Protein expression yields of 8-12 mg/L of culture, corresponding to about two thirds of the expression level of the wild-type DHFR protein, are sufficient to provide fluorinated proteins suitable for 19F-NMR spectroscopy and other sample-intensive methods. The use of a nonessential "21st" tRNA/synthetase pair will permit incorporation of a wide range of analogues, once the synthetase specificity has been modified accordingly.
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PMID:Expansion of the genetic code: site-directed p-fluoro-phenylalanine incorporation in Escherichia coli. 952 Nov 19

The 4-pentenoyl group and a number of derivatives have been studied as protecting groups for N(alpha) of the aminoacyl moiety in misacylated tRNAs. The unsubstituted 4-pentenoyl group itself was found to function as efficiently as any of the derivatives studied. Four different N-(4-pentenoyl)aminoacyl-tRNA(CUA)s were prepared and shown to undergo deprotection readily upon admixture of aqueous iodine; the derived misacylated tRNAs all functioned well as suppressors of a nonsense codon in an in vitro protein biosynthesizing system. Also prepared were four N(alpha)-(4-pentenoyl)aspartyl-tRNA(CUA)s that were protected on the side chain carboxylate as the nitroveratryl ester. Following treatment with aqueous iodine, the misacylated suppressor tRNAs incorporated the aspartate derivatives into position 27 of dihydrofolate reductase by suppression of a UAG codon in the mRNA. The suppression yields were significantly better than those obtained when side chain protection was absent. The resulting "caged proteins" were inactive, but full catalytic potential was restored by irradiation under conditions sufficient to effect deprotection of the side chain carboxylate moiety.
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PMID:Misacylated Transfer RNAs Having a Chemically Removable Protecting Group. 1167 75

By employing a general biosynthetic method for the elaboration of proteins containing unnatural amino acid analogues, we incorporated (aminooxy)acetic acid into positions 10 and 27 of Escherichia coli dihydrofolate reductase. Introduction of the modified amino acid into DHFR was accomplished in an in vitro protein biosynthesizing system by readthrough of a nonsense (UAG) codon with a suppressor tRNA that had been activated with (aminooxy)acetic acid. Incorporation of the amino acid proceeded with reasonable efficiency at codon position 10 but less well at position 27. (Aminooxy)acetic acid was also incorporated into position 72 of DNA polymerase beta. Peptides containing (aminooxy)acetic acid have been shown to adopt a preferred conformation involving an eight-membered ring that resembles a gamma-turn. Accordingly, the present study may facilitate the elaboration of proteins containing conformationally biased peptidomimetic motifs at predetermined sites. The present results further extend the examples of ribosomally mediated formation of peptide bond analogues of altered connectivity and provide a conformationally biased linkage at a predetermined site. It has also been shown that the elaborated protein can be cleaved chemically at the site containing the modified amino acid.
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PMID:Site-specific incorporation of (aminooxy)acetic acid into proteins. 1223 90

Extensive studies aiming to establish the structure and root of the Eukaryota tree by phylogenetic analyses of molecular sequences have thus far not resulted in a generally accepted tree. To re-examine the eukaryotic phylogeny using alternative genes, and to obtain a more robust inference for the root of the tree as well as the relationship among major eukaryotic groups, we sequenced the genes encoding isoleucyl-tRNA and valyl-tRNA synthetases, cytosolic-type heat shock protein 90, and the largest subunit of RNA polymerase II from several protists. Combined maximum likelihood analyses of 22 protein-coding genes including the above four genes clearly demonstrated that Diplomonadida and Parabasala shared a common ancestor in the rooted tree of Eukaryota, but only when the fast-evolving sites were excluded from the original data sets. The combined analyses, together with recent findings on the distribution of a fused dihydrofolate reductase-thymidylate synthetase gene, narrowed the possible position of the root of the Eukaryota tree on the branch leading to Opisthokonta or to the common ancestor of Diplomonadida/Parabasala. However, the analyses did not agree with the position of the root located on the common ancestor of Opisthokonta and Amoebozoa, which was argued by Stechmann and Cavalier-Smith [Curr. Biol. 13:R665-666, 2003] based on the presence or absence of a three-gene fusion of the pyrimidine biosynthetic pathway: carbamoyl-phosphate synthetase II, dihydroorotase, and aspartate carbamoyltransferase. The presence of the three-gene fusion recently found in the Cyanidioschyzon merolae (Rhodophyta) genome sequence data supported our analyses against the Stechmann and Cavalier-Smith-rooting in 2003.
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PMID:Root of the Eukaryota tree as inferred from combined maximum likelihood analyses of multiple molecular sequence data. 1549 53

Introduction of a yeast suppressor tRNA (ytRNA(Phe)(CUA)) and a mutant yeast phenylalanyl-tRNA synthetase (yPheRS (T415G)) into an Escherichia coli expression host allows in vivo incorporation of phenylalanine analogues into recombinant proteins in response to amber stop codons. However, high-fidelity incorporation of non-natural amino acids is precluded in this system by mischarging of ytRNA(Phe)(CUA) with tryptophan (Trp) and lysine (Lys). Here we show that ytRNA(Phe)(CUA) and yPheRS can be redesigned to achieve high-fidelity amber codon suppression through delivery of p-bromophenylalanine (pBrF). Two strategies were used to reduce misincorporation of Trp and Lys. First, Lys misincorporation was eliminated by disruption of a Watson-Crick base pair between nucleotides 30 and 40 in ytRNA(Phe)(CUA). Loss of this base pair reduces mischarging by the E. coli lysyl-tRNA synthetase. Second, the binding site of yPheRS was redesigned to enhance specificity for pBrF. Specifically, we used the T415A variant, which exhibits 5-fold higher activity toward pBrF as compared to Trp in ATP-PP(i) exchange assays. Combining mutant ytRNA(Phe)(CUA) and yPheRS (T415A) allowed incorporation of pBrF into murine dihydrofolate reductase in response to an amber codon with at least 98% fidelity.
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PMID:Design of a bacterial host for site-specific incorporation of p-bromophenylalanine into recombinant proteins. 1695 16

In the presence of Phe-SA, the stable sulfamoyl analogue of phenylalanyl adenylate, the codon (UUU/UUC) for phenylalanine (Phe) can be reassigned to naphthylalanine (Nap) bound to tRNA(Phe). The efficiency and selectivity of this Phe-to-Nap reassignment induced by the "orthogonal reacylation stalling" method was demonstrated at the single-codon level in the translation of mRNAs of dihydrofolate reductase (DHFR) and a 24-mer oligopeptide. In the prokaryotic translation system with essential preincubation, the endogenous precharged phenylalanyl-tRNA(Phe) undergoes deacylation and reacylation of the resulting tRNA(Phe) is inhibited by the action of Phe-SA to kill the phenylalanyl-tRNA synthetase activity. The significance of the present small-molecule-based approach to sense-codon templated natural-unnatural peptides is discussed.
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PMID:A simple approach to sense codon-templated synthesis of natural/unnatural hybrid peptides. 1715 Jul 35

Combination of nonsense suppression and protein-ribosome-mRNA (PRM) complexation techniques leads to a new strategy "read-through polysome/ribosome display", which is designed to display full-length open reading frame (ORF) domain of the protein on the natural mRNA templates. The optimised conditions are to use the anticodon-adjusted tRNA for Leu as a nonsense suppressor in a reconstituted translation system containing diminished amounts of release factors (RFs). When applied to pseudo-natural mRNAs of Escherichia coli dihydrofolate reductase (E. coli DHFR), the input mRNA was recovered as a polysome complex displaying full-length DHFR.
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PMID:In vitro read-through polysome/ribosome display of full-length protein ORF and it's applications. 1715 Jul 36


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