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D Söll

Publications and source records attributed to D Söll.

At least 73 records · Page 4Linked to original sources
PubMed1996

Genetic analysis of functional connectivity between substrate recognition domains of Escherichia coli glutaminyl-tRNA synthetase.

It has previously been shown that the single mutation E222K in glutaminyl-tRNA synthetase (GlnRS) confers a temperature-sensitive phenotype on Escherichia coli. Here we report the isolation of a pseudorevertant of this mutation, E222K/C171G, which was subsequently employed to investigate the role of these residues in substrate discrimination. The three-dimensional structure of the tRNA(Gln): GlnRS: ATP ternary complex revealed that both E222 and C171 are close to regions of the protein involved in interactions with both the acceptor stem and the 3' end of tRNA(Gln). The potential involvement of E222 and C171 in these interactions was confirmed by the observation that GlnRS-E222K was able to mischarge supF tRNA(Tyr) considerably more efficiently than the wild-type enzyme, whereas GlnRS-E222K/C171G could not. These differences in substrate specificity also extended to anticodon recognition, with the double mutant able to distinguish supE tRNA(CUA)(Gln) from tRNA2(Gln) considerably more efficiently than GlnRS E222K. Furthermore, GlnRS-E222K was found to have a 15-fold higher K(m) for glutamine than the wild-type enzyme, whereas the double mutant only showed a 7-fold increase. These results indicate that the C171G mutation improves both substrate discrimination and recognition at three domains in GlnRS-E222K, confirming recent proposals that there are extensive interactions between the active site and regions of the enzyme involved in tRNA binding.

Adenosine Triphosphate↗
PubMed1996

Asn-tRNA in Lactobacillus bulgaricus is formed by asparaginylation of tRNA and not by transamidation of Asp-tRNA.

In many organisms (e.g., gram-positive eubacteria) Gin-tRNA is not formed by direct glutaminylation of tRNAGln but by a specific transamidation of Glu-tRNAGln. We wondered whether a similar transamidation pathway also operates in the formation of Asn-tRNA in these organisms. Therefore we tested in S-100 preparations of Lactobacillus bulgaricus, a gram-positive eubacterium, for the conversion by an amidotransferase of [14C]Asp-tRNA to [14C]Asn-tRNA. As no transamidation was observed, we searched for genes for asparaginyl-tRNA synthetase (AsnRS). Two DNA fragments (from different locations of the L.bulgaricus chromosome) were found each containing an ORF whose sequence resembled that of the Escherichia coli asnS gene. The derived amino acid sequences of the two ORFs (432 amino acids) were the same and 41% identical with E.coli AsnRS. When one of the ORFs was expressed in E.coli, it complemented the temperature sensitivity of an E.coli asnS mutant. S-100 preparations of this transformant showed increased charging of unfractionated L.bulgaricus tRNA with asparagine. Deletion of the 3'-terminal region of the L.bulgaricus AsnRS gene led to loss of its complementation and aminoacylation properties. This indicates that L.bulgaricus contains a functional AsnRS. Thus, the transamidation pathway operates only for Gin-tRNAGln formation in this organism, and possibly in all gram-positive eubacteria.

Amino Acid Sequence↗
PubMed1996

Interactions between tRNA identity nucleotides and their recognition sites in glutaminyl-tRNA synthetase determine the cognate amino acid affinity of the enzyme.

Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs both ensure accurate RNA recognition and prevent the binding of noncognate substrates. Here we show for Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) that the accuracy of tRNA recognition also determines the efficiency of cognate amino acid recognition. Steady-state kinetics revealed that interactions between tRNA identity nucleotides and their recognition sites in the enzyme modulate the amino acid affinity of GlnRS. Perturbation of any of the protein-RNA interactions through mutation of either component led to considerable changes in glutamine affinity with the most marked effects seen at the discriminator base, the 10:25 base pair, and the anticodon. Reexamination of the identity set of tRNA(Gln) in the light of these results indicates that its constituents can be differentiated based upon biochemical function and their contribution to the apparent Gibbs' free energy of tRNA binding. Interactions with the acceptor stem act as strong determinants of tRNA specificity, with the discriminator base positioning the 3' end. The 10:25 base pair and U35 are apparently the major binding sites to GlnRS, with G36 contributing both to binding and recognition. Furthermore, we show that E. coli tryptophanyl-tRNA synthetase also displays tRNA-dependent changes in tryptophan affinity when charging a noncognate tRNA. The ability of tRNA to optimize amino acid recognition reveals a novel mechanism for maintaining translational fidelity and also provides a strong basis for the coevolution of tRNAs and their cognate synthetases.

Amino Acyl-tRNA Synthetases↗
PubMed1996

A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis.

The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.

Amino Acid Sequence↗
PubMed1996

Transfer RNA-dependent cognate amino acid recognition by an aminoacyl-tRNA synthetase.

An investigation of the role of tRNA in the catalysis of aminoacylation of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) has revealed that the accuracy of specific interactions between GlnRS and tRNAGln determines amino acid affinity. Mutations in GlnRS at D235, which makes contacts with nucleotides in the acceptor stem of tRNAGln, and at R260 in the enzyme's active site were found to be independent during tRNA binding but interactive for aminoacylation. Characterization of mutants of GlnRS at position 235, showed amino acid recognition to be tRNA mediated. Aminoacylation of tRNA(CUA)Tyr [tyrT (UAG)] by GlnRS-D235H resulted in a 4-fold increase in the Km for the Gln, which was reduced to a 2-fold increase when A73 was replaced with G73. These and previous results suggest that specific interactions between GlnRS and tRNAGln ensure the accurate positioning of the 3' terminus. Disruption of these interactions can change the Km for Gln over a 30-fold range, indicating that the accuracy of aminoacylation is regulated by tRNA at the level of both substrate recognition and catalysis. The observed role of RNA as a cofactor in optimizing amino acid activation suggests that the tRNAGln-GlnRS complex may be partly analogous to ribonucleoprotein enzymes where protein-RNA interactions facilitate catalysis.

Adenosine Triphosphate↗
PubMed1996

Functional connectivity between tRNA binding domains in glutaminyl-tRNA synthetase.

The structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) in complex with tRNAGln and ATP has identified a number a sequence-specific protein-tRNA interactions. The contribution to glutamine identity has previously been determined for the nucleotides in tRNAGln. Here, we report the mutational analysis of residues in all three tRNA recognition domains of GlnRS, thus completing a survey of the major sequence-specific contacts between GlnRS and tRNAGln. Specifically, we analyzed the GlnRS determinants involved in recognition of the anticodon which is essential for glutamine identity and in the communication of anticodon recognition to the acceptor binding domain in GlnRS. A combined in vivo and in vitro approach has demonstrated that Arg341, which makes a single sequence-specific hydrogen bond with U35 in the anticodon of tRNAGln, is involved in initial RNA recognition and is an important positive determinant for this base in both cognate and non- cognate tRNA contexts. However, Arg341, as well as Arg402, which interacts with G36 in the anticodon, are negative determinants for non-cognate nucleotides at their respective positions. Analysis of acceptor-anticodon binding double mutants and of a mutation of Glu323 in the loop-strand-helix connectivity subdomain in GlnRS has further implicated this domain in the functional communication of anticodon recognition. The better than expected activity (anticooperativity) of these double mutants has led us to propose an "anticodon-independent" mechanism, in which the removal of certain synthetase interactions with the anticodon eliminates structural constraints, thus allowing the relaxed specificity mutants in the acceptor binding domain ot make more productive interactions.

Amino Acyl-tRNA Synthetases↗
PubMed1996

The C-terminal extension of yeast seryl-tRNA synthetase affects stability of the enzyme and its substrate affinity.

Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) contains a 20-amino acid C-terminal extension, which is not found in prokaryotic SerRS enzymes. A truncated yeast SES1 gene, lacking the 60 base pairs that encode this C-terminal domain, is able to complement a yeast SES1 null allele strain; thus, the C-terminal extension in SerRS is dispensable for the viability of the cell. However, the removal of the C-terminal peptide affects both stability of the enzyme and its affinity for the substrates. The truncation mutant binds tRNA with 3.6-fold higher affinity, while the Km for serine is 4-fold increased relative to the wild-type SerRS. This indicates the importance of the C-terminal extension in maintaining the overall structure of SerRS.

Amino Acid Sequence↗
PubMed1996

Aminoacyl-tRNA synthetases optimize both cognate tRNA recognition and discrimination against noncognate tRNAs.

Specific protein--nucleic acid interactions are usually the product of sequence-dependent hydrogen bonding. However, in the crystal structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) in complex with tRNAGln, leucine 136 (Leu136) stabilizes the disruption of the weak first (U1-A72) base pair in tRNAGln by stacking between A72 and G2. We have demonstrated, by a combined in vivo and in vitro mutational analysis, that Leu136 is important for tRNA specificity despite making no hydrogen bonds with tRNAGln. Both more (L136F) and less (L136V, L136M, L136A, and L136T) mischarging mutants of GlnRS have been identified. GlnRS(L136F) is more mischarging and less specific than wild-type GlnRS in vivo, due not to an increased affinity for the noncognate tRNAs but to a decreased affinity for tRNAGln. Also, unlike other mischarging mutants of GlnRS that have been characterized, it does not exhibit generally relaxed tRNA specificity in vivo and mischarges only a subset of the tRNAs tested. A possible sequence preference for a Py1-Pu72/Pu2-Py71 combination is suggested. The L136A/M/T/V mutants are the first GlnRS variants, including wild-type, expressed on pBR322 which no longer mischarge tyrT(UAG) in vivo. We have shown that, while the L136A mutant is less mischarging than wild-type both in vivo and in vitro, it is not more specific as it also exhibits reduced affinity for its cognate glutamine tRNA. On the basis of these results, we suggest that the aminoacyl-tRNA synthetases have evolved to balance cognate tRNA recognition and discrimination against noncognate tRNAs.

Base Sequence↗
PubMed1996

Escherichia coli tryptophanyl-tRNA synthetase mutants selected for tryptophan auxotrophy implicate the dimer interface in optimizing amino acid binding.

Tryptophan auxotrophs of Escherichia coli in which mutations were mapped to the trpS locus (encoding tryptophanyl-tRNA synthetase) have been previously isolated. We have investigated the tryptophanyl-tRNA synthetase (TrpRS) purified from six auxotrophic strains for changes in amino acid activation and aminoacylation. Steady-state kinetic analyses show that these mutant TrpRS proteins have increases in the apparent KM for tryptophan, decreases in turnover number, or both, without significant changes in the apparent KM for ATP or tRNA(Trp). The crystal structure of a highly homologous tryptophanyl-tRNA synthetase from Bacillus stearothermophilus in a complex with the cognate aminoacyl adenylate allowed us to place the mutations in a structural context. The mutations in the enzymes are located in the KMSKS loop (M196I), in or near the active site (D112E, P129S, A133E) or far from the active site. The last three mutants (T60R, L91F, G329S) could not be predicted by examination of the protein structure as they line an interface between the C-terminal alpha-helix of one subunit and the Rossmann folds of both subunits, thus affecting a specific region of the dimer interface. These results support a role for dimerization in properly configuring the two active sites of the dimeric enzymes in the Trp/Tyr subclass of class I aminoacyl-tRNA synthetases in order to achieve optimal catalysis.

Amino Acid Sequence↗
PubMed1996

A second and differentially expressed glutamyl-tRNA reductase gene from Arabidopsis thaliana.

5-Aminolevulinic acid (ALA) is the universal precursor of tetrapyrroles (e.g., chlorophylls and hemes). In the chloroplasts of plants and in several eubacterial species ALA is formed in a two-step process known as the C5 pathway. In the first step, glutamyl-tRNA reductase (GluTR), converts glutamate of glutamyl-tRNA to glutamate 1-semialdehyde (GSA) which is rearranged to ALA by glutamate 1-semialdehyde-2,1-aminomutase (GSA-A) in the second step. Since ALA formation is a limiting step in chlorophyll biosynthesis, GluTR, which is encoded by the HEMA gene in Arabidopsis thaliana plays a vital role in that biosynthesis. Here we report the occurrence of a second functional HEMA gene (HEMA2) in A. thaliana. This gene was isolated by screening a genomic library with a probe from HEMA1. The nucleotide sequence of the cDNA and the corresponding genomic DNA indicates that the Arabidopsis HEMA2 gene contains two short introns (285 bp and 159 bp). The deduced amino acid sequence predicts a HEMA2 protein of 530 amino acids with 79% identity to the HEMA1-encoded GluTR. The 5'-flanking sequence of the HEMA2 gene includes several motifs (e.g., GT-1 boxes, GATA motifs) similar to light-responsive regulatory elements found in light-inducible genes. Unlike the HEMA1 transcript, which is present in all parts of the plant, HEMA2 is expressed in low levels in roots and flowers. The presence of a second functional HEMA gene in Arabidopsis raises the possibility that two C5 pathways exist in chloroplasts.

Aldehyde Oxidoreductases↗
PubMed1996

Homologous expression and purification of mutants of an essential protein by reverse epitope-tagging.

Purification of mutant enzymes is a prime requirement of biophysical and biochemical studies. Our investigations on the essential Escherichia coli enzyme glutaminyl-tRNA synthetase demand mutant enzymes free of any wild-type protein contamination. However, as it is not possible to express noncomplementing mutant enzymes in an E. coli glnS-deletion strain, we developed a novel strategy to address these problems. Instead of following the common tactic of epitope-tagging the mutant protein of interest on an extrachromosomal genetic element, we fused a reporter epitope to the 5' end of the chromosomal glnS-gene copy: this is referred to as 'reverse epitope-tagging.' The corresponding strain, E. coli HAPPY101, displays a normal phenotype, and glutaminyl-tRNA synthetase is exclusively present as an epitope-tagged form in cell-free extracts. Here we report the use of E. coli HAPPY101 to express and purify a number of mutant glutaminyl-tRNA synthetases independently of their enzymatic activity. In this process, epitope-tagged wild-type protein is readily separated from mutant enzymes by conventional chromatographic methods. In addition, the absence of wild-type can be monitored by immunodetection using a monoclonal antibody specific for the epitope. The strategy described here for expression and purification of an essential enzyme is not restricted to glutaminyl-tRNA synthetase and should be applicable to any essential enzyme that retains sufficient activity to sustain growth following reverse epitope-tagging.

Amino Acyl-tRNA Synthetases↗
PubMed1996

Glutaminyl-tRNA synthetase: from genetics to molecular recognition.

Accurately aminoacylated tRNAs are an a priori requirement for translation of the genetic code. They are synthesized by the aminoacyl-tRNA synthetases which select both the correct amino acid and tRNA from a total of more than 400 possible combinations. Genetic, biochemical and structural studies have begun to reveal the mechanisms by which this specificity is achieved by Escherichia coli glutaminyl-tRNA synthetase (GlnRS). Sequence-specific interactions between GlnRS and tRNA(Gln) determine both the accuracy of tRNA selection and the efficiency of aminoacylation. Thus, amino acid recognition is tRNA-dependent. Consequently, while a noncognate tRNA may be recognized by GlnRS, the resulting tRNA-enzyme complex displays a considerably reduced affinity for glutamine compared to wild-type. This mechanism now provides a ready explanation as to why the majority of tRNA mischarging events, including those originally described over 25 years ago for GlnRS, impair cellular viability only to a limited degree.

Amino Acyl-tRNA Synthetases↗
PubMed1996

Lactobacillus bulgaricus asparagine synthetase and asparaginyl-tRNA synthetase: coregulation by transcription antitermination?

Genes encoding the ammonia-dependent asparagine synthetase (asnA) and asparaginyl-tRNA synthetase (asnS) have been cloned from Lactobacillus bulgaricus ATCC 11842. The nucleotide sequence suggests that asnA and asnS are organized as one operon and regulated by the tRNA-directed transcription antitermination mechanism (T. M. Henkin, Mol. Microbiol. 13:381-387, 1994).

Amino Acid Sequence↗
PubMed1995

A broadly applicable continuous spectrophotometric assay for measuring aminoacyl-tRNA synthetase activity.

We describe a convenient, simple and novel continuous spectrophotometric method for the determination of aminoacyl-tRNA synthetase activity. The assay relies upon the measurement of inorganic pyrophosphate generated in the first step of the aminoacylation of a tRNA. Pyrophosphate release is coupled to inorganic pyrophosphatase, to generate phosphate, which in turn is used as the substrate of purine nucleoside phosphorylase to catalyze the N-glycosidic cleavage of 2-amino 6-mercapto 7-methylpurine ribonucleoside. Of the reaction products, ribose 1-phosphate and 2-amino 6-mercapto 7-methylpurine, the latter has a high absorbance at 360 nm relative to the nucleoside and hence provides a spectrophotometric signal that can be continuously followed. The non-destructive nature of the spectrophotometric assay allowed the re-use of the tRNAs in question in successive experiments. The usefulness of this method was demonstrated for glutaminyl-tRNA synthetase (GlnRS) and tryptophanyl-tRNA synthetase. Initial velocities measured using this assay correlate closely with those assayed by quantitation of [3H]Gln-tRNA or [14C]Trp-tRNA formation respectively. In both cases amino acid transfer from the aminoacyl adenylate to the tRNA represents the rate determining step. In addition, aminoacyl adenylate formation by aspartyl-tRNA synthetase was followed and provided a more sensitive means of active site titration than existing techniques. Finally, this novel method was used to provide direct evidence for the cooperativity of tRNA and ATP binding to GlnRS.

Acylation↗
PubMed1995

Divergence of glutamate and glutamine aminoacylation pathways: providing the evolutionary rationale for mischarging.

Aminoacyl-tRNA for protein synthesis is produced through the action of a family of enzymes called aminoacyl-tRNA synthetases. A general rule is that there is one aminoacyl-tRNA synthetase for each of the standard 20 amino acids found in all cells. This is not universal, however, as a majority of prokaryotic organisms and eukaryotic organelles lack the enzyme glutaminyl-tRNA synthetase, which is responsible for forming Gln-tRNAGln in eukaryotes and in Gram-negative eubacteria. Instead, in organisms lacking glutaminyl-tRNA synthetase, Gln-tRNAGln is provided by misacylation of tRNAGln with glutamate by glutamyl-tRNA synthetase, followed by the conversion of tRNA-bound glutamate to glutamine by the enzyme Glu-tRNAGln amidotransferase. The fact that two different pathways exist for charging glutamine tRNA indicates that ancestral prokaryotic and eukaryotic organisms evolved different cellular mechanisms for incorporating glutamine into proteins. Here, we explore the basis for diverging pathways for aminoacylation of glutamine tRNA. We propose that stable retention of glutaminyl-tRNA synthetase in prokaryotic organisms following a horizontal gene transfer event from eukaryotic organisms (Lamour et al. 1994) was dependent on the evolving pool of glutamate and glutamine tRNAs in the organisms that acquired glutaminyl-tRNA synthetase by this mechanism. This model also addresses several unusual aspects of aminoacylation by glutamyl- and glutaminyl-tRNA synthetases that have been observed.

Acylation↗