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D Söll

Publications and source records attributed to D Söll.

At least 55 records · Page 3Linked to original sources
PubMed1998

Glutamyl-tRNA(Gln) amidotransferase in Deinococcus radiodurans may be confined to asparagine biosynthesis.

Asparaginyl-tRNA (Asn-tRNA) and glutaminyl-tRNA (Gln-tRNA) are essential components of protein synthesis. They can be formed by direct acylation by asparaginyl-tRNA synthetase (AsnRS) or glutaminyl-tRNA synthetase (GlnRS). The alternative route involves transamidation of incorrectly charged tRNA. Examination of the preliminary genomic sequence of the radiation-resistant bacterium Deinococcus radiodurans suggests the presence of both direct and indirect routes of Asn-tRNA and Gln-tRNA formation. Biochemical experiments demonstrate the presence of AsnRS and GlnRS, as well as glutamyl-tRNA synthetase (GluRS), a discriminating and a nondiscriminating aspartyl-tRNA synthetase (AspRS). Moreover, both Gln-tRNA and Asn-tRNA transamidation activities are present. Surprisingly, they are catalyzed by a single enzyme encoded by three ORFs orthologous to Bacillus subtilis gatCAB. However, the transamidation route to Gln-tRNA formation is idled by the inability of the discriminating D. radiodurans GluRS to produce the required mischarged Glu-tRNAGln substrate. The presence of apparently redundant complete routes to Asn-tRNA formation, combined with the absence from the D. radiodurans genome of genes encoding tRNA-independent asparagine synthetase and the lack of this enzyme in D. radiodurans extracts, suggests that the gatCAB genes may be responsible for biosynthesis of asparagine in this asparagine prototroph.

Acylation↗
PubMed1998

Major identity element of glutamine tRNAs from Bacillus subtilis and Escherichia coli in the reaction with B. subtilis glutamyl-tRNA synthetase.

Early investigations revealed that Bacillus subtilis glutamyl-tRNA synthetase [GluRS (bs)] is responsible for aminoacylating both glutamate tRNA [tRNA(Glu) (bs)] and glutamine tRNA [tRNA(Gln) (bs)] with glutamate. The same Bacillus enzyme can also efficiently attach glutamate to one isoacceptor glutamine tRNA [tRNA(Gln) (ec)] of Escherichia coli in vitro but not to tRNA2(Gln) (ec) and tRNA(Glu) (ec). To characterize identity elements of these glutamine tRNAs in the interaction with GluRS (bs), tRNA2(Gln) (ec), tRNA1(Gln) (ec), three other mutant glutamine tRNAs [tRNA2(Gln) (AU) (C34 --> U34), tRNA2(Gln) (12M) (C34 --> U34, 31A-U39 --> 31U-A39), and tRNA2(Gln) (M21) (64C --> G50 --> 64G-C50, 63U-A51 --> 63A-U51)] originated from tRNA2(Gln) (ec), tRNA(Gln) (bs), and a mutant tRNAM(Gln) (bs) whose U at the 34th position (U34), was replaced to C (C34), were produced in E. coli. All of the E. coli glutamine tRNAs containing U34 such as tRNA1(Gln), tRNA2(Gln) (AU), and tRNA2(Gln) (12M) could be charged with glutamate by GluRS (bs), whereas tRNA2(Gln) (ec) and its T-stem mutant tRNA2(Gln) (M21) containing C34 could not be charged by the same enzyme. The unique change of C34 to U34 of tRNA2(Gln) (ec) acquired glutamate acceptor activity by GluRS (bs). This result suggests that the U34 is the major identity element of tRNA1(Gln) (ec) in the recognition by GluRS (bs). The same situation was found in tRNA(Gln) (bs). The glutamate acceptor activity of tRNA(Gln) (bs) disappeared on replacement of U34 to C34. To find out whether modified bases in tRNA(Gln) (bs) are involved in the recognition by GluRS (bs), glutamylation of tRNA(Gln) (bs) produced by in vitro transcription was also examined but the in vitro transcript of tRNA(Gln) (bs) could not be charged with glutamic acid by GluRS (bs). All of these mean that the major recognition element for GluRS (bs) is U at the 34th position of both tRNA(Gln) (bs) and tRNA1(Gln) (ec) as a modified form.

Amino Acyl-tRNA Synthetases↗
PubMed1998

Retracing the evolution of amino acid specificity in glutaminyl-tRNA synthetase.

Molecular phylogenetic studies of glutaminyl-tRNA synthetase suggest that it has relatively recently evolved from the closely related enzyme glutamyl-tRNA synthetase. We have now attempted to retrace one of the key steps in this process by selecting glutaminyl-tRNA synthetase mutants displaying enhanced glutamic acid recognition. Mutagenesis of two residues proximal to the active site, Phe-90 and Tyr-240, was found to improve glutamic acid recognition 3-5-fold in vitro and resulted in the misacylation of tRNA(Gln) with glutamic acid. In vivo expression of the genes encoding these misacylating variants of glutaminyl-tRNA synthetase reduced cellular growth rates by 40%, probably as a result of an increase in translational error rates. These results provide the first biochemical evidence that glutaminyl-tRNA synthetase originated through duplication and consequent diversification of an ancestral glutamyl-tRNA synthetase-encoding gene.

Amino Acid Sequence↗
PubMed1998

The terminal adenosine of tRNA(Gln) mediates tRNA-dependent amino acid recognition by glutaminyl-tRNA synthetase.

Sequence-specific interactions between Escherichia coli glutaminyl-tRNA synthetase and tRNA(Gln) have been shown to determine the apparent affinity of the enzyme for its cognate amino acid glutamine during aminoacylation. Specifically, structural and biochemical studies suggested that residues Asp66, Tyr211, and Phe233 in glutaminyl-tRNA synthetase could potentially facilitate cognate amino recognition through their specific interactions with both A76 of tRNA(Gln)++ and glutamine. These residues were randomly mutated and the resulting glutaminyl-tRNA synthetase variants were screened in vivo for changes in their ability to recognize noncognate tRNAs and retention of tRNA-glutaminylation activity. When the variants selected in this way were characterized in vitro, they all showed dramatic decreases in apparent affinity (KM) for glutamine but little or no change in cognate tRNA affinity. Conservative replacements such as Y211F, F233L, and D66E resulted in 60-, 19-, and 18-fold increases compared to wild-type in the KM for glutamine, respectively, but had little effect on the turnover number (kcat). Nonconservative replacements affected both KM for glutamine and kcat; Y211S, F233D, and D66F displayed 1700, 3700, and 1200-fold decreases in kcat/KM for glutamine compared to wild-type. Double mutant cycle analysis indicated that Tyr211, and Phe233 interact strongly to enhance glutamine binding. These data now show that Asp66, Tyr211 and Phe233 mediate tRNA-dependent cognate amino acid recognition via the invariant 3'-terminal adenosine of tRNA(Gln).

Adenosine↗
PubMed1998

Maize mitochondrial seryl-tRNA synthetase recognizes Escherichia coli tRNA(Ser) in vivo and in vitro.

In our studies to analyze the structure/function relationships among cytoplasmic and organellar seryl-tRNA synthetases (SerRS), we have characterized a Zea mays cDNA (SerZMm) encoding a protein with significant similarity to prokaryotic SerRS enzymes. To demonstrate the functional identity of SerZMm, the gene sequence encoding the putative mature protein was cloned. This construct complemented in vivo a temperature-sensitive Escherichia coli serS mutant strain. The mature SerZMm protein overexpressed in Escherichia coli efficiently aminoacylated bacterial tRNA(Ser) in vitro, while yeast tRNA was a poor substrate. These data identify SerZMm as an organellar maize seryl-tRNA synthetase, the first plant organellar SerRS to be cloned. The analysis of its N-terminal targeting signal suggests a mitochondrial function for the SerZMm protein in maize.

Amino Acid Sequence↗
PubMed1998

Root-growth behavior of the Arabidopsis mutant rgr1. Roles of gravitropism and circumnutation in the waving/coiling phenomenon.

In this study we investigated the kinetics of the gravitropic response of the Arabidopsis mutant rgr1 (reduced root gravitropism). Although the rate of curvature in rgr1, which is allelic to axr4, was smaller than in the wild type (ecotype Wassilewskija), curvature was initiated in the same region of the root, the distal elongation zone. The time lag for the response was unaffected in the mutant; however, the gravitropic response of rgr1 contained a feature not found in the wild type: when roots growing along the surface of an agar plate were gravistimulated, there was often an upward curvature that initiated in the central elongation zone. Because this response was dependent on the tactile environment of the root, it most likely resulted from the superposition of the waving/coiling phenomenon onto the gravitropic response. We found that the frequency of the waving pattern and circumnutation, a cyclic endogenous pattern of root growth, was the same in rgr1 and in the wild type, so the waving/coiling phenomenon is likely governed by circumnutation patterns. The amplitudes of these oscillations may then be selectively amplified by tactile stimulation to provide a directional preference to the slanting.

Arabidopsis↗
PubMed1998

Sequence divergence of seryl-tRNA synthetases in archaea.

The genomic sequences of Methanococcus jannaschii and Methanobacterium thermoautotrophicum contain a structurally uncommon seryl-tRNA synthetase (SerRS) sequence and lack an open reading frame (ORF) for the canonical cysteinyl-tRNA synthetase (CysRS). Therefore, it is not clear if Cys-tRNACys is formed by direct aminoacylation or by a transformation of serine misacylated to tRNACys. To address this question, we prepared SerRS from two methanogenic archaea and measured the enzymatic properties of these proteins. SerRS was purified from M. thermoautotrophicum; its N-terminal peptide sequence matched the sequence deduced from the relevant ORF in the genomic data of M. thermoautotrophicum and M. jannaschii. In addition, SerRS was expressed from a cloned Methanococcus maripaludis serS gene. The two enzymes charged serine to their homologous tRNAs and also accepted Escherichia coli tRNA as substrate for aminoacylation. Gel shift experiments showed that M. thermoautotrophicum SerRS did not mischarge tRNACys with serine. This indicates that Cys-tRNACys is formed by direct acylation in these organisms.

Amino Acid Sequence↗
PubMed1997

Archaeal-type lysyl-tRNA synthetase in the Lyme disease spirochete Borrelia burgdorferi.

Lysyl-tRNAs are essential for protein biosynthesis by ribosomal mRNA translation in all organisms. They are synthesized by lysyl-tRNA synthetases (EC 6.1.1.6), a group of enzymes composed of two unrelated families. In bacteria and eukarya, all known lysyl-tRNA synthetases are subclass IIc-type aminoacyl-tRNA synthetases, whereas some archaea have been shown to contain an unrelated class I-type lysyl-tRNA synthetase. Examination of the preliminary genomic sequence of the bacterial pathogen Borrelia burgdorferi, the causative agent of Lyme disease, indicated the presence of an open reading frame with over 55% similarity at the amino acid level to archaeal class I-type lysyl-tRNA synthetases. In contrast, no coding region with significant similarity to any class II-type lysyl-tRNA synthetase could be detected. Heterologous expression of this open reading frame in Escherichia coli led to the production of a protein with canonical lysyl-tRNA synthetase activity in vitro. Analysis of B. burgdorferi mRNA showed that the lysyl-tRNA synthetase-encoding gene is highly expressed, confirming that B. burgdorferi contains a functional class I-type lysyl-tRNA synthetase. The detection of an archaeal-type lysyl-tRNA synthetase in B. burgdorferi and other pathogenic spirochetes, but not to date elsewhere in bacteria or eukarya, indicates that the gene that encodes this enzyme has a common origin with its orthologue from the archaeal kingdom. This difference between the lysyl-tRNA synthetases of spirochetes and their hosts may be readily exploitable for the development of anti-spirochete therapeutics.

Amino Acid Sequence↗
PubMed1997

A euryarchaeal lysyl-tRNA synthetase: resemblance to class I synthetases.

The sequencing of euryarchaeal genomes has suggested that the essential protein lysyl-transfer RNA (tRNA) synthetase (LysRS) is absent from such organisms. However, a single 62-kilodalton protein with canonical LysRS activity was purified from Methanococcus maripaludis, and the gene that encodes this protein was cloned. The predicted amino acid sequence of M. maripaludis LysRS is similar to open reading frames of unassigned function in both Methanobacterium thermoautotrophicum and Methanococcus jannaschii but is unrelated to canonical LysRS proteins reported in eubacteria, eukaryotes, and the crenarchaeote Sulfolobus solfataricus. The presence of amino acid motifs characteristic of the Rossmann dinucleotide-binding domain identifies M. maripaludis LysRS as a class I aminoacyl-tRNA synthetase, in contrast to the known examples of this enzyme, which are class II synthetases. These data question the concept that the classification of aminoacyl-tRNA synthetases does not vary throughout living systems.

Acylation↗
PubMed1997

Glu-tRNAGln amidotransferase: a novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation.

The three genes, gatC, gatA, and gatB, which constitute the transcriptional unit of the Bacillus subtilis glutamyl-tRNAGln amidotransferase have been cloned. Expression of this transcriptional unit results in the production of a heterotrimeric protein that has been purified to homogeneity. The enzyme furnishes a means for formation of correctly charged Gln-tRNAGln through the transamidation of misacylated Glu-tRNAGln, functionally replacing the lack of glutaminyl-tRNA synthetase activity in Gram-positive eubacteria, cyanobacteria, Archaea, and organelles. Disruption of this operon is lethal. This demonstrates that transamidation is the only pathway to Gln-tRNAGln in B. subtilis and that glutamyl-tRNAGln amidotransferase is a novel and essential component of the translational apparatus.

Acylation↗
PubMed1997

Defining the active site of yeast seryl-tRNA synthetase. Mutations in motif 2 loop residues affect tRNA-dependent amino acid recognition.

The active site of class II aminoacyl-tRNA synthetases contains the motif 2 loop, which is involved in binding of ATP, amino acid, and the acceptor end of tRNA. In order to characterize the active site of Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS), we performed in vitro mutagenesis of the portion of the SES1 gene encoding the motif 2 loop. Substitutions of amino acids conserved in the motif 2 loop of seryl-tRNA synthetases from other sources led to loss of complementation of a yeast SES1 null allele strain by the mutant yeast SES1 genes. Steady-state kinetic analyses of the purified mutant SerRS proteins revealed elevated Km values for serine and ATP, accompanied by decreases in kcat (as expected for replacement of residues involved in aminoacyl-adenylate formation). The differences in the affinities for serine and ATP, in the absence and presence of tRNA are consistent with the proposed conformational changes induced by positioning the 3'-end of tRNA into the active site, as observed recently in structural studies of Thermus thermophilus SerRS (Cusack, S., Yaremchuk, A., and Tukalo, M. (1996) EMBO J. 15, 2834-2842). The crystal structure of this moderately homologous prokaryotic counterpart of the yeast enzyme allowed us to produce a model of the yeast SerRS structure and to place the mutations in a structural context. In conjunction with structural data for T. thermophilus SerRS, the kinetic data presented here suggest that yeast seryl-tRNA synthetase displays tRNA-dependent amino acid recognition.

Amino Acid Sequence↗
PubMed1997

Aminoacyl-tRNA synthesis: divergent routes to a common goal.

Aminoacyl-tRNAs are key components in protein synthesis. They are formed directly by correct acylation of tRNA (by aminoacyl-tRNA synthetases) or indirectly by tRNA-dependent transformation of misacylated tRNAs. The accuracy of aminoacyl-tRNA synthesis is enhanced by a number of further protein-RNA or protein-protein interactions, some of which are restricted to Archaea, and might reflect adaptation mechanisms to diverse conditions.

Amino Acyl-tRNA Synthetases↗
PubMed1997

A nuclear genetic lesion affecting Saccharomyces cerevisiae mitochondrial translation is complemented by a homologous Bacillus gene.

A novel Bacillus gene was isolated and characterized. It encodes a homolog of Saccharomyces cerevisiae Pet112p, a protein that has no characterized relative and is dispensable for cell viability but required for mitochondrial translation. Expression of the Bacillus protein in yeast, modified to ensure mitochondrial targeting, partially complemented the phenotype of the pet112-1 mutation, demonstrating a high degree of evolutionary conservation for this as yet unidentified component of translation.

Bacillus subtilis↗
PubMed1997

Glutaminyl-tRNA synthetase.

Among the twenty aminoacyl-tRNA synthetases glutaminyl-tRNA synthetase occupies a special position: it is one of only two enzymes of this family which is not found in all organisms, being mainly absent from gram positive eubacteria, archaebacteria and organelles. The E. coli GlnRS is relatively small with 553 amino acids and a molecular mass of 64.4 kDa and functions as a monomer. The mammalian enzymes are somewhat larger and can be parts of multienzyme complexes. Crystal structures were solved of E. coli GlnRS complexed with tRNA(Gln) and ATP, of this complex containing tRNA(Gln) replaced by unmodified tRNA(Gln), and of three complexes with mutated GlnRS enzymes. The GlnRS molecule consists of four domains, the catalytic site is located in the Rossman fold, typical for class I synthetases, and the reaction mechanism follows the normal adenylate pathway. The enzyme shows many similarities with glutamyl-tRNA synthetase; a common ancestor of both molecules is well established. In the E. coli system recognition of the cognate tRNA has been studied in many details using both natural and artificial mutants of tRNA(Gln) and of the enzyme: GlnRS recognizes mainly conventional parts of the tRNA molecule, namely some bases of the anticodon loop and parts of the acceptor stem.

Adenosine Triphosphate↗
PubMed1997

Glutamyl-tRNA sythetase.

Glutamyl-tRNA synthetase (GluRS) belongs to the class I aminoacyl-tRNA synthetases and shows several similarities with glutaminyl-tRNA synthetase concerning structure and catalytic properties. Phylogenetic studies suggested that both diverged from an ancestral glutamyl-tRNA synthetase responsible for the gluta-mylation of tRNA(Glu) and tRNA(Gln), and whose Glu-tRNA(Gln) product is transformed into Gln-tRNA(Gln) by a specific amidotransferase. This pathway is present in gram-positive and some gram-negative eubacteria, in some archae and in organelles, and was never found jointly with a glutaminyl-tRNA synthetase. Other gram-negative eubacteria and the cytoplasm of eukaryotes contain a glutamyl-tRNA synthetase specific for tRNA(Glu), and a glutaminyl-tRNA synthetase. Bacterial glutamyl-tRNA synthetases consist of about 500 amino acid residues, possess molecular masses of about 50 kDa, and act as monomers. In higher eukaryotes chimeric glutamyl-prolyl-tRNA synthetases were found, in a high molecular mass complex containing several other aminoacyl-tRNA synthetases. To date one crystal structure of a glutamyl-tRNA synthetase (Thermus thermophilus) has been solved. The molecule has the form of a bent cylinder and consists of four domains. The N-terminal half (domains 1 and 2) contains the 'Rossman fold' typical for class I synthetases and resembles the corresponding part of E. coli GlnRS, whereas the C-terminal half exhibits a GluRS-specific structure. As found for the other aminoacyl-tRNA synthetases the catalytic pathway of GluRS includes the formation of an aminoacyl adenylate in the first reaction step, but GluRS shares a special property with GlnRS and ArgRS: the ATP/PPi pyrophosphate exchange reaction is only catalyzed in the presence of the cognate tRNA. Compared with other aminoacyl-tRNA synthetases a relatively high number of investigations deals with recognition of tRNA(Glu) by GluRS. Besides interactions between the enzyme and the acceptor stem and the anticodon of tRNA(Glu), checking of the dihydrouridine arm and of the variable loop by GluRS are documented.

Acylation↗
PubMed1997

tRNA-dependent amino acid transformations.

Aminoacyl-tRNAs are either synthesized directly by aminoacyl-tRNA synthetases or indirectly by tRNA-dependent transformation of mischarged tRNAs. The enzymes which participate in the indirect routes may be interesting targets in the development of novel therapeutic compounds. We have purified one such enzyme, Glu-tRNA(Gln) amidotransferase from Bacillus subtilis, to homogeneity and present the initial biochemical characterization data.

Bacillus subtilis↗
PubMed1997

Aminoacyl-tRNA synthesis in Archaea.

The mechanism of aminoacyl-tRNA synthesis differs substantially between Archaea, Bacteria and Eukarya. Sequencing of archaeal genomes has suggested that the asparaginyl-, cysteinyl-, glutaminyl- and lysyl-tRNA synthetases are absent from a number of organisms in this kingdom. The absence of the asparaginyl- and glutaminyl-tRNA synthetases is in agreement with the observation that Asn-tRNA and Gln-tRNA are synthesized by tRNA-dependent transamidation of Asp-tRNA and Glu-tRNA respectively in the archaeon Haloferax volcanii. Biochemical and genetic studies have now shown that while the cysteinyl- and lysyl-tRNA synthetases are present, the enzymes responsible for these activities are unique to Archaea.

Amino Acyl-tRNA Synthetases↗