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D Söll

Publications and source records attributed to D Söll.

At least 127 records · Page 7Linked to original sources
PubMed1991

Anticodon and acceptor stem nucleotides in tRNA(Gln) are major recognition elements for E. coli glutaminyl-tRNA synthetase.

The correct attachment of amino acids to their corresponding (cognate) transfer RNA catalysed by aminoacyl-tRNA synthetases is a key factor in ensuring the fidelity of protein biosynthesis. Previous studies have demonstrated that the interaction of Escherichia coli tRNA(Gln) with glutaminyl-tRNA synthetase (GlnRS) provides an excellent system to study this highly specific recognition process, also referred to as 'tRNA identity'. Accurate acylation of tRNA depends mainly on two principles: a set of nucleotides in the tRNA molecule (identity elements) responsible for proper discrimination by aminoacyl-tRNA synthetases and competition between different synthetases for tRNAs. Elements of glutamine identity are located in the anticodon and in the acceptor stem region, including the discriminator base. We report here the production of more than 20 tRNA(2Gln) mutants at positions likely to be involved in tRNA discrimination by the enzyme. Unmodified tRNA, containing the wild-type anticodon and U or G at its 5'-terminus, can be aminocylated by GlnRS with similar kinetic parameters to native tRNA(2Gln). By in vitro aminoacylation the mutant tRNAs showed decreases of up to 3 x 10(5)-fold in the specificity constant (kcat/KM)14 with the major contribution of kcat. Despite these large changes, some of these mutant tRNAs are efficient amber suppressors in vivo. Our results show that strong elements for glutamine identity reside in the anticodon region and in positions 2 and 3 of the acceptor stem, and that the contribution of different identity elements to the overall discrimination varies significantly. We discuss our data in the light of the crystal structure of the GlnRS:tRNA(Gln) complex.

Amino Acyl-tRNA Synthetases↗
PubMed1991

Two glutamyl-tRNA reductase activities in Escherichia coli.

delta-Aminolevulinic acid (ALA) is the first committed precursor for tetrapyrrole biosynthesis. ALA formation in Escherichia coli occurs in a tRNA-dependent three-step conversion from glutamate. Glu-tRNA reductase is the key enzyme in this pathway. E. coli K12 contains two Glu-tRNA reductase activities which differ in their molecular weights. Here we describe the purification of one of these enzymes. Four different chromatographic separations yielded a nearly homogeneous protein. Its apparent molecular mass under denaturing (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and nondenaturing conditions (rate zonal sedimentation and gel filtration) is 85,000 +/- 5,000 Da. This indicates a monomeric structure for the active enzyme. Gel filtration and glycerol gradient centrifugation indicate that the other activity has a molecular mass of 45,000 +/- 5,000 Da. In the presence of NADPH both enzyme activities converted E. coli Glu-tRNA(2Glu) to glutamate 1-semialdehyde. Addition of GTP or hemin did not affect the reductase activity. Both enzymes display sequence-specific recognition of tRNA; E. coli Glu-tRNA(2Glu) is a good substrate while the Chlamydomonas reinhardtii, Bacillus subtilis, and Synechocystis Glu-tRNA(Glu) species are poorly recognized.

Aminolevulinic Acid↗
PubMed1991

Purification and functional characterization of glutamate-1-semialdehyde aminotransferase from Chlamydomonas reinhardtii.

The formation of delta-aminolevulinic acid, the first committed precursor of chlorophyll biosynthesis, occurs in the chloroplast of plants and algae by the C5-pathway, a three-step, tRNA-dependent transformation of glutamate. Previously, we reported the purification and characterization of the first two enzymes of this pathway, glutamyl-tRNA synthetase and Glu-tRNA reductase from the green alga Chlamydomonas reinhardtii (Chen, M.-W., Jahn, D., Schön, A., O'Neill, G. P., and Söll, D. (1990) J. Biol. Chem. 265, 4054-4057 and Chen, M.-W., Jahn, D., O'Neill, G. P., and Söll, D. (1990) J. Biol. Chem. 265, 4058-4063). Here we present the purification of the third enzyme of the pathway, the glutamate-1-semialdehyde aminotransferase from C. reinhardtii. The enzyme was purified from the membrane fraction of a whole cell extract employing four different chromatographic separations. The apparent molecular mass of the protein was approximately 43,000 Da as analyzed by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by nondenaturing rate zonal sedimentation on glycerol gradients, and by gel filtration. By these criteria, the enzyme in its active form is a monomer of 43,000 Da. In the presence of pyridoxal 5'-phosphate, purified glutamate-1-semialdehyde aminotransferase converts synthetic glutamate 1-semialdehyde to delta-aminolevulinic acid. The enzyme is inhibited by gabaculine and aminooxyacetate, both typical inhibitors of aminotransferases. The purified glutamate-1-semialdehyde aminotransferase successfully reconstitutes the whole C5-pathway in vitro from glutamate in the presence of purified glutamyl-tRNA synthetase, glutamyl-tRNA reductase, Mg2+, ATP, NADPH, tRNA, and pyridoxal 5'-phosphate.

Aminooxyacetic Acid↗
PubMed1991

Mutant enzymes and tRNAs as probes of the glutaminyl-tRNA synthetase: tRNA(Gln) interaction.

This paper focuses on several aspects of the specificity of mutants of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) and tRNA(Gln). Temperature-sensitive mutants located in glnS, the gene for GlnRS, have been described previously. The mutations responsible for the temperature-sensitive phenotype were analyzed, and pseudorevertants of these mutants isolated and characterized. The nature of these mutations is discussed in terms of their location in the three-dimensional structure of the tRNA(Gln).GlnRS complex. In order to characterize the specificity of the aminoacylation reaction, mutant tRNA(Gln) species were synthesized with either a 2'-deoxy AMP or 3'-deoxy AMP as their 3'-terminal nucleotide. Subsequent assays for aminoacylation and ATP/PPi exchange activity established the esterification of glutamine to the 2'-hydroxyl of the terminal adenosine; there is no glutaminylation of the 3'-OH group. This correlates with the classification of GlnRS as a class I aminoacyl-tRNA synthetase. Mutations in tRNA(Gln) are discussed which affect the recognition of GlnRS and the current concept of glutamine identity in E coli is reviewed.

Base Sequence↗
PubMed1991

The Human Genome Project: a paradigm for information management in the life sciences.

The major product of the Human Genome Project will be a series of linked data sets containing the genetic and physical location of all genes on each chromosome, plus the complete nucleotide sequence of the genome for humans and several model organisms. Here we summarize the current status of attempts to collect, analyze, and distribute this information in an electronically accessible form. Although formidable problems remain to be solved in the acquisition and adequate representation of the genetic, physical, and biological data, this project is a model for the rapid dissemination of genome and related information in biology and medicine.

Base Sequence↗
PubMed1991

delta-Aminolevulinic acid dehydratase deficiency can cause delta-aminolevulinate auxotrophy in Escherichia coli.

Ethylmethane sulfonate-induced mutants of several Escherichia coli strains that required delta-aminolevulinic acid (ALA) for growth were isolated by penicillin enrichment or by selection for respiratory-defective strains resistant to the aminoglycoside antibiotic kanamycin. Three classes of mutants were obtained. Two-thirds of the strains were mutants in hemA. Representative of a third of the mutations was the hem-201 mutation. This mutation was mapped to min 8.6 to 8.7. Complementation of the auxotrophic phenotype by wild-type DNA from the corresponding phage 8F10 allowed the isolation of the gene. DNA sequence analysis revealed that the hem-201 gene encoded ALA dehydratase and was similar to a known hemB gene of E. coli. Complementation studies of hem-201 and hemB1 mutant strains with various hem-201 gene subfragments showed that hem-201 and the previously reported hemB1 mutation are in the same gene and that no other gene is required to complement the hem-201 mutant. ALA-forming activity from glutamate could not be detected by in vitro or in vivo assays. Extracts of hem-201 cells had drastically reduced ALA dehydratase levels, while cells transformed with the plasmid-encoded wild-type gene possessed highly elevated enzyme levels. The ALA requirement for growth, the lack of any ALA-forming enzymatic activity, and greatly reduced ALA dehydratase activity of the hem-201 strain suggest that a diffusible product of an enzyme in the heme biosynthetic pathway after ALA formation is involved in positive regulation of ALA biosynthesis. In contrast to the hem-201 mutant, previously isolated hemB mutants were not ALA auxotrophs and had no detectable ALA dehydratase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminolevulinic Acid↗
PubMed1991

The Escherichia coli hemL gene encodes glutamate 1-semialdehyde aminotransferase.

delta-Aminolevulinic acid (ALA), the first committed precursor of porphyrin biosynthesis, is formed in Escherichia coli by the C5 pathway in a three-step, tRNA-dependent transformation from glutamate. The first two enzymes of this pathway, glutamyl-tRNA synthetase and Glu-tRNA reductase, are known in E. coli (J. Lapointe and D. Söll, J. Biol. Chem. 247:4966-4974, 1972; D. Jahn, U. Michelsen, and D. Söll, J. Biol. Chem. 266:2542-2548, 1991). Here we present the mapping and cloning of the gene for the third enzyme, glutamate 1-semialdehyde (GSA) aminotransferase, and an initial characterization of the purified enzyme. Ethylmethane sulfonate-induced mutants of E. coli AB354 which required ALA for growth were isolated by selection for respiration-defective strains resistant to the aminoglycoside antibiotic kanamycin. Two mutations were mapped to min 4 at a locus named hemL. Map positions and resulting phenotypes suggest that hemL may be identical with the earlier described porphyrin biosynthesis mutation popC. Complementation of the auxotrophic phenotype by wild-type DNA from the corresponding clone pLC4-43 of the Clarke-Carbon bank (L. Clarke and J. Carbon, Cell 9:91-99, 1976) allowed the isolation of the gene. Physical mapping showed that hemL mapped clockwise next to fhuB. The hemL gene product was overexpressed and purified to apparent homogeneity. The pure protein efficiently converted GSA to ALA. The reaction was stimulated by the addition of pyridoxal 5' -phosphate or pyridoxamine 5' -phosphate and inhibited by gabaculine or aminooxyacetic acid. The molecular mass of the purified GSA aminotransferase under denaturing conditions was 40,000 Da, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has apparent native molecular mass of approximately 80,000 Da, as determined by rate zonal sedimentation on glycerol gradients and molecular sieving through Superose 12, which indicates a homodimeric alpha2, structure of the protein.

Aminolevulinic Acid↗
PubMed1990

The accuracy of aminoacylation--ensuring the fidelity of the genetic code.

The fidelity of protein biosynthesis rests not only on the proper interaction of the messenger RNA codon with the anticodon of the tRNA, but also on the correct attachment of amino acids to their corresponding (cognate) transfer RNA (tRNA) species. This process is catalyzed by the aminoacyl-tRNA synthetases which discriminate with remarkable selectivity amongst many structurally similar tRNAs. The basis for this highly specific recognition of tRNA by these enzymes (also referred to as 'tRNA identity') is currently being elucidated by genetic, biochemical and biophysical techniques. At least two factors are important in determining the accuracy of aminoacylation: a) 'identity elements' in tRNA denote nucleotides in certain positions crucial for protein interactions determining specificity, and b) the occurrence in vivo of competition between synthetases for a particular tRNA which may have ambiguous identity.

Amino Acyl-tRNA Synthetases↗
PubMed1990

The RNA component of RNase P in Schizosaccharomyces species.

In the fission yeast Schizosaccharomyces pombe, the enzyme RNAse P copurifies with two RNAs, K1- and K2-RNA, which are identical except for their termini [1] and which are encoded by a single gene [2]. We have undertaken the cloning of the K-RNA genes in related organisms in order to gain comparative structural information. Because the K-RNA sequence is poorly conserved across species, we have cloned the K-RNA genes in the Schizosaccharomyces species S. malidevorans, S. japonicus, S. versatilis, and S. octosporus. Of the 4 species, only S. octosporus contains a K-RNA gene different from that in S. pombe; the gene diverges by 20%. Based on the two sequences, nuclease protection data and computer analysis, we have proposed a secondary structure model for the K-RNA. Northern analysis shows the K-RNA genes in all four Schizosaccharomyces species to be expressed as two RNAs, as in S. pombe.

Base Sequence↗
PubMed1990

Purification and functional characterization of the Glu-tRNA(Gln) amidotransferase from Chlamydomonas reinhardtii.

The formation of glutaminyl-tRNA (Gln-tRNA) in Bacilli, chloroplasts, and mitochondria occurs in a two-step reaction. This involves misacylation of tRNA(Gln) with glutamate by glutamyl-tRNA synthetase and subsequent amidation of Glu-tRNA(Gln) to the correctly acylated Gln-tRNA(Gln) by a specific amidotransferase (Schön, A., Kannangara, C. G., Gough, S., and Söll, D. (1988) Nature 331, 187-190). Here we demonstrate the existence of this pathway in green algae and describe the purification of the Glu-tRNA(Gln) amidotransferase from Chlamydomonas reinhardtii. The purified enzyme showed an Mr of approximately 120,000 when analyzed by glycerol gradient sedimentation and gel filtration. An apparent Mr of 63,000 of the denatured protein was demonstrated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. This indicates that the enzyme possesses an alpha 2 structure. The substrate for the purified enzyme is Glu-tRNA(Gln) but not Glu-tRNA(Glu). The enzyme requires ATP, Mg2+, and an amide donor for the conversion. Acceptable amide donors are glutamine, asparagine, and ammonia. Blocking of the glutamine-dependent reaction by alkylation of the protein with 6-diazo-5-oxonorleucine did not inhibit the ammonia-dependent reaction, suggesting that the enzyme has separate glutamine and ammonia binding sites. As suggested by Wilcox (Wilcox, M. (1969) Eur. J. Biochem. 11, 405-412) the amidation reaction may involve glutamyl-phosphate formation, since ATP is cleaved to ADP when the enzyme is incubated with Glu-tRNA(Gln) and ATP. In common with other glutamine amidotransferases, the enzyme also possesses low glutaminase activity. The purified Glu-tRNA(Gln) amidotransferase forms a stable complex with Glu-tRNA(Gln) in the presence of ATP and Mg2+ but in the absence of the amide donor as determined by gradient centrifugation.

Adenosine Triphosphate↗
PubMed1990

Purification and characterization of Chlamydomonas reinhardtii chloroplast glutamyl-tRNA synthetase, a natural misacylating enzyme.

Glutamyl-tRNA synthetase from Chlamydomonas reinhardtii was purified by sequential column chromatography on DEAE-cellulose, phosphocellulose, Mono Q, and Mono S. The apparent molecular mass of the protein when analyzed under both denaturing conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and nondenaturing conditions (rate zonal sedimentation on glycerol gradients) was 62,000 Da; this indicates that the active enzyme is a monomer. The purified glutamyl-tRNA synthetase was identified as the chloroplast enzyme by its tRNA charging specificity. Reversed-phase chromatography of unfractionated C. reinhardtii tRNA resolved four peaks of glutamate acceptor RNA when assayed with the purified enzyme. The enzyme can also glutamylate Escherichia coli tRNA(2Glu), but not cytoplasmic tRNA(Glu) from yeast or barley. In addition, the enzyme misacylates chloroplast tRNA(Gln) with glutamate. A similar mischarging phenomenon has been demonstrated for the barley chloroplast enzyme (Schön, A., Kannangara, C.G., Gough, S., and Söll, D. (1988) Nature 331, 187-190) and for Bacillus subtilis glutamyl-tRNA synthetase (Proulx, M., Duplain, L., Lacoste, L., Yaguchi, M., and Lapointe, J. (1983) J. Biol. Chem. 258, 753-759).

Acylation↗
PubMed1990

Purification of the glutamyl-tRNA reductase from Chlamydomonas reinhardtii involved in delta-aminolevulinic acid formation during chlorophyll biosynthesis.

The formation of delta-aminolevulinic acid, the first committed precursor in porphyrin biosynthesis, occurs in certain bacteria and in the chloroplasts of plants and algae in a three-step, tRNA-dependent transformation of glutamate. Glutamyl-tRNA reductase, the second enzyme of this pathway, reduces the activated carboxyl group of glutamyl-tRNA (Glu-tRNA) in the presence of NADPH and releases glutamate 1-semialdehyde (GSA). We have purified Glu-tRNA reductase from Chlamydomonas reinhardtii by employing six different chromatographic separations. The apparent molecular mass of the protein when analyzed under both denaturing (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and nondenaturing conditions (rate zonal sedimentation on glycerol gradients) was 130,000 Da; this indicates that the active enzyme is a monomer. In the presence of NADPH Glu-tRNA reductase catalyzed the reduction to GSA of glutamate acylated to the homologous tRNA. Thus, the reductase alone is sufficient for conversion of Glu-tRNA to GSA. In the absence of NADPH, a stable complex of Glu-tRNA reductase with Glu-tRNA can be isolated.

Aminolevulinic Acid↗