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)

The discriminator nucleotide (position 73) in tRNA has long been thought to play a role in tRNA identity as it is the only variable single-stranded nucleotide that is found near the site of aminoacylation. For this reason, a complete mutagenic analysis of the discriminator in three Escherichia coli amber suppressor tRNA backgrounds was undertaken; supE and supE-G1C72 glutamine tRNAs, gluA glutamate tRNA and supF tyrosine tRNA. The effect of mutation of the discriminator base on the identity of these tRNAs in vivo was assayed by N-terminal protein sequencing of E. coli dihydrofolate reductase, which is the product of suppression by the mutated amber suppressors, and confirmed by amino acid specific suppression experiments. In addition, suppressor efficiency assays were used to estimate the efficiency of aminoacylation in vivo. Our results indicate that the supE glutamine tRNA context can tolerate multiple mutations (including mutation of the discriminator and first base-pair) and still remain predominantly glutamine-accepting. Discriminator mutants of gluA glutamate tRNA exhibit increased and altered specificity probably due to the reduced ability of other synthetases to compete with glutamyl-tRNA synthetase. In the course of these experiments, a glutamate-specific mutant amber suppressor, gluA-A73, was created. Finally, in the case of supF tyrosine tRNA, the discriminator is an important identity element with partial to complete loss of tyrosine specificity resulting from mutation at this position. It is clear from these experiments that it may not be possible to assign a specific role in tRNA identity to the discriminator. The identity of a tRNA in vivo is determined by competition among aminoacyl-tRNA synthetases, which is in turn modulated by the nucleotide substitution as well as the tRNA context.
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PMID:Synthetase competition and tRNA context determine the in vivo identify of tRNA discriminator mutants. 147 77

The middle base (U35) of the anticodon of tRNA(Gln) is a major element ensuring the accuracy of aminoacylation by Escherichia coli glutaminyl-tRNA synthetase (GlnRS). An opal suppressor of tRNA(Gln) (su+2UGA) containing C35 (anticodon UCA) was isolated by genetic selection and mutagenesis. Suppression of a UGA mutation in the E. coli fol gene followed by N-terminal sequence analysis of purified dihydrofolate reductase showed that this tRNA was an efficient suppressor that inserted predominantly tryptophan. Mutations of the 3-70 base pair (U70 and A3U70) were made. These mutants of su+2UGA are less efficient suppressors and inserted predominantly tryptophan in vivo; alanine insertion was not observed. Mutations of the discriminator nucleotide (A73, U73, C73) result in very weak opal suppressors. Aminoacylation in vitro by E. coli TrpRS of tRNA(Gln) transcripts mutated in the anticodon demonstrate that TrpRS recognizes all three nucleotides of the anticodon. The results show the interchangeability of the glutamine and tryptophan identities by base substitutions in their respective tRNAs. The amber suppressor (anticodon CUA) tRNA(Trp) was known previously to insert predominantly glutamine. We show that the opal suppressor (anticodon UCA) tRNA(Gln) inserts mainly tryptophan. Discrimination by these synthetases for tRNA includes position 35, with recognition of C35 by TrpRS and U35 by GlnRS. As the use of the UGA codon as tryptophan in mycoplasma and in yeast mitochondria is conserved, recognition of the UCA anticodon by TrpRS may also be maintained in evolution.
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PMID:Switching tRNA(Gln) identity from glutamine to tryptophan. 156 39

Splicing of tRNA precursors in extracts of Saccharomyces cerevisiae requires the action of two enzymes: a site specific endonuclease and a tRNA ligase. The tRNA ligase contains three distinct enzymatic activities: a polynucleotide kinase, a cyclic phosphodiesterase, and an RNA ligase. The polypeptide also has a high affinity pre-tRNA binding site based on its ability to form stable complexes with pre-tRNA substrates. To investigate the organization of functional enzymatic and binding elements within the polypeptide a series of defined tRNA ligase gene deletions were constructed and corresponding proteins were expressed in Escherichia coli as fusions with bacterial dihydrofolate reductase (DHFR). The DHFR/ligase derivative proteins were then efficiently purified by affinity chromatography. The complete ligase fusion protein retained enzymatic and binding activities which were unaffected by the presence of the DHFR segment. Examination of tRNA ligase deletion derivatives revealed that the amino-terminal region was required for adenylylation, while the carboxyl-terminal region was sufficient for cyclic phosphodiesterase activity. Deletions within the central region affected kinase activity. Pre-tRNA binding activity was not strictly correlated with a distinct enzymatic domain. A DHFR/ligase-derived protein lacking kinase activity efficiently joined tRNA halves. We postulate that this variant utilizes a novel RNA ligation mechanism.
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PMID:Deletion analysis of a multifunctional yeast tRNA ligase polypeptide. Identification of essential and dispensable functional domains. 185 Apr 8

Using synthetic oligonucleotides, we have constructed a collection of Escherichia coli amber suppressor tRNA genes. In order to determine their specificities, these tRNAs were each used to suppress an amber (UAG) nonsense mutation in the E. coli dihydrofolate reductase gene fol. The mutant proteins were purified and subjected to N-terminal sequence analysis to determine which amino acid had been inserted by the suppressor tRNAs at the position of the amber codon. The suppressors can be classified into three groups on the basis of the protein sequence information. Class I suppressors, tRNA(CUAAla2), tRNA(CUAGly1), tRNA(CUAHisA), tRNA(CUALys) and tRNA(CUAProH), inserted the predicted amino acid. The class II suppressors, tRNA(CUAGluA), tRNA(CUAGly2) and tRNA(CUAIle1) were either partially or predominantly mischarged by the glutamine aminoacyl tRNA synthetase. The class III suppressors, tRNA(CUAArg), tRNA(CUAAspM), tRNA(CUAIle2), tRNA(CUAThr2), tRNA(CUAMet(m)) and tRNA(CUAVal) inserted predominantly lysine.
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PMID:Construction of Escherichia coli amber suppressor tRNA genes. III. Determination of tRNA specificity. 214 50

Fusions between the TRM1 gene of Saccharomyces cerevisiae and COXIV or DHFR were made to examine the mitochondrial targeting signals of N2,N2-dimethylguanosine-specific tRNA methyltransferase [tRNA (m2(2)G)dimethyltransferase]. This enzyme is responsible for the modification of both mitochondrial and cytoplasmic tRNAs. We have previously shown that two forms of the enzyme are translated from two in-frame ATGs in this gene, that they differ by a 16-amino-acid amino-terminal extension, and that both the long and short forms are imported into mitochondria. Results of studies to test the ability of various TRM1 sequences to serve as surrogate mitochondrial targeting signals for passenger protein import in vitro and in vivo showed that the most efficient signal derived from tRNA (m2(2)G)dimethyltransferase included a combination of sequences from both the amino-terminal extension and the amino terminus of the shorter form of the enzyme. The amino-terminal extension itself did not serve as an independent mitochondrial targeting signal, whereas the amino terminus of the shorter form of tRNA (m2(2)G)dimethyltransferase did function in this regard, albeit inefficiently. We analyzed the first 48 amino acids of tRNA (m2(2)G)dimethyltransferase for elements of primary and secondary structure shared with other known mitochondrial targeting signals. The results lead us to propose that the most efficient signal spans the area around the second ATG of TRM1 and is consistent with the idea that there is a mitochondrial targeting signal present at the amino terminus of the shorter form of the enzyme and that the amino-terminal extension augments this signal by extending it to form a larger, more efficient mitochondrial targeting signal.
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PMID:Amino-terminal extension generated from an upstream AUG codon increases the efficiency of mitochondrial import of yeast N2,N2-dimethylguanosine-specific tRNA methyltransferases. 265

FokI, a class-IIS restriction endonuclease, cleaves double-stranded DNA to produce a protruding 5' end consisting of four nucleotides, 10-13 residues 3' from the nonpalindromic recognition sequence, GGATG. Cassettes which utilize this separation of cleavage and recognition site have been constructed for the purpose of linker mutagenesis and DNA replacement experiments. The cassettes are flanked by FokI recognition sequences oriented such that the FokI cleavage sites are several nucleotides beyond the cassette/vector fusion sites. FokI excises the cassette and several base pairs of the neighboring vector sequence. The ends produced in the vector by FokI cleavage are generally noncomplementary and suitable for the insertion of a segment of synthesized double-stranded replacement DNA. A cassette which contains a tyrosine tRNA suppressor gene (supF) is selectable by the suppression of amber mutations in the recipient host. A vector containing a pBR322-derived origin of replication, the Escherichia coli xanthine-guanine phosphoribosyl transferase gene as a selectable marker, and no FokI sites has been constructed for use with the FokI cassettes. An experiment which utilized the FokI/supF cassette to modify the N-terminal coding region of the R388 dihydrofolate reductase gene is described.
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PMID:The use of a selectable FokI cassette in DNA replacement mutagenesis of the R388 dihydrofolate reductase gene. 282 Aug 44

Initiation factor eIF2 binds GTP and promotes the binding of methionyl-tRNA to ribosomes. Biochemical and sequence evidence suggests that the GTP might bind to either the beta- or gamma-subunit of eIF2. Mutations were made in the NKXD consensus elements found in both subunits and individual mutant forms were overexpressed in transiently transfected COS-1 cells. The effect on the translational efficiency of a reporter mRNA for dihydrofolate reductase was monitored. Mutations in the gamma-subunit cause severe repression of protein synthesis, whereas those in the beta-subunit are only mildly inhibitory. The results support the view that GTP binds exclusively to the gamma-subunit.
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PMID:Mutations in the NKXD consensus element indicate that GTP binds to the gamma-subunit of translation initiation factor eIF2. 755 78

MOD5, a gene responsible for the modification of A37 to isopentenyl A37 of both cytosolic and mitochondrial tRNAs, encodes two isozymes. Initiation of translation at the first AUG of the MOD5 open reading frame generates delta 2-isopentenyl pyrophosphate:tRNA isopentanyl transferase I (IPPT-I), which is located predominantly, but not exclusively, in the mitochondria. Initiation of translation at a second AUG generates IPPT-II, which modifies cytoplasmic tRNA. IPPT-II is unable to target to mitochondria. The N-terminal sequence present in IPPT-I and absent in IPPT-II is therefore necessary for mitochondrial targeting. In these studies, we fused MOD5 sequences encoding N-terminal regions to genes encoding passenger proteins, pseudomature COXIV and dihydrofolate reductase, and studied the ability of these chimeric proteins to be imported into mitochondria both in vivo and in vitro. We found that the sequences necessary for mitochondrial import, amino acids 1 to 11, are not sufficient for efficient mitochondrial targeting and that at least some of the amino acids shared by IPPT-I and IPPT-II comprise part of the mitochondrial targeting information. We used indirect immunofluorescence and cell fractionation to locate the MOD5 isozymes in yeast. IPPT-I was found in two subcellular compartments: mitochondria and the cytosol. We also found that IPPT-II had two subcellular locations: nuclei and the cytosol. The nuclear location of this protein is surprising because the A37-->isopentenyl A37 modification had been predicted to occur in the cytoplasm. MOD5 is one of the first genes reported to encode isozymes found in three subcellular compartments.
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PMID:Subcellular locations of MOD5 proteins: mapping of sequences sufficient for targeting to mitochondria and demonstration that mitochondrial and nuclear isoforms commingle in the cytosol. 813 35

N-substituted diphenimides and 6,7-dihydro-5H-dibenz[c,e]azepines demonstrated significant cytotoxic activity against the growth of murine and human cells. These derivatives were active against leukemias, carcinomas and sarcomas. Different derivatives with N-substitutions showed specific activity against the growth of several tumor types. These agents inhibited L1210 leukemia IMP dehydrogenase and PRPP amido transferase activities; this was reflected in the inhibition of purine and DNA synthesis. Other sites inhibited to a minor degree by these agents included DNA polymerase alpha, r- and tRNA polymerases, ribonucleoside reductase, dihydrofolate reductase, pyrimidine synthesis, and nucleoside kinase. d(NTP) pool levels were reduced after 24 h incubation with these derivatives. L1210 DNA strand scission was evident after drug treatment.
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PMID:The cytotoxicity of N-substituted diphenimides and 6,7-dihydro-5H-dibenz[c,e]azepines. 829 66

The use of synthetic tRNA for in vitro protein engineering was tested in a coupled transcription/translation system prepared from Escherichia coli. DNA sequences similar to the natural tRNA(Ala/UGC) gene from E. coli but with different anticodons were synthesized in vitro, cloned into a DNA plasmid, and then transcribed in vitro with T7 RNA polymerase. The UGC alanine anticodon was changed to CUA corresponding to the UAG stop codon, CCU corresponding to the rarely used AGG arginine codon, and two four-nucleotide anticodons used to suppress stop codons. Bacterial dihydrofolate reductase was the test protein. Its cloned coding sequence was mutagenized at the GUG codon for valine-75 to correspond to the anticodons of the tRNA constructs, and then the plasmids were used to direct the synthesis of dihydrofolate reductase in the coupled transcription/translation system containing the corresponding synthetic tRNA. The results indicate that all four synthetic tRNAs were functionally active in the synthesis of full-length, enzymatically active dihydrofolate reductase protein.
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PMID:In vitro protein engineering using synthetic tRNA(Ala) with different anticodons. 834 99


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