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D Söll

Publications and source records attributed to D Söll.

At least 37 records · Page 2Linked to original sources
PubMed2000

Domain-specific recruitment of amide amino acids for protein synthesis.

The formation of aminoacyl-transfer RNA is a crucial step in ensuring the accuracy of protein synthesis. Despite the central importance of this process in all living organisms, it remains unknown how archaea and some bacteria synthesize Asn-tRNA and Gln-tRNA. These amide aminoacyl-tRNAs can be formed by the direct acylation of tRNA, catalysed by asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase, respectively. A separate, indirect pathway involves the formation of mis-acylated Asp-tRNA(Asn) or Glu-tRNA(Gln), and the subsequent amidation of these amino acids while they are bound to tRNA, which is catalysed by amidotransferases. Here we show that all archaea possess an archaea-specific heterodimeric amidotransferase (encoded by gatD and gatE) for Gln-tRNA formation. However, Asn-tRNA synthesis in archaea is divergent: some archaea use asparaginyl-tRNA synthetase, whereas others use a heterotrimeric amidotransferase (encoded by the gatA, gatB and gatC genes). Because bacteria primarily use transamidation, and the eukaryal cytoplasm uses glutaminyl-tRNA synthetase, it appears that the three domains use different mechanisms for Gln-tRNA synthesis; as such, this is the only known step in protein synthesis where all three domains have diverged. Closer inspection of the two amidotransferases reveals that each of them recruited a metabolic enzyme to aid its function; this provides direct evidence for a relationship between amino-acid metabolism and protein biosynthesis.

Amides↗
PubMed2000

The heterotrimeric Thermus thermophilus Asp-tRNA(Asn) amidotransferase can also generate Gln-tRNA(Gln).

Thermus thermophilus strain HB8 is known to have a heterodimeric aspartyl-tRNA(Asn) amidotransferase (Asp-AdT) capable of forming Asn-tRNA(Asn) [Becker, H.D. and Kern, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12832-12837]. Here we show that, like other bacteria, T. thermophilus possesses the canonical set of amidotransferase (AdT) genes (gatA, gatB and gatC). We cloned and sequenced these genes, and constructed an artificial operon for overexpression in Escherichia coli of the thermophilic holoenzyme. The overproduced T. thermophilus AdT can generate Gln-tRNA(Gln) as well as Asn-tRNA(Asn). Thus, the T. thermophilus tRNA-dependent AdT is a dual-specific Asp/Glu-AdT resembling other bacterial AdTs. In addition, we observed that removal of the 44 carboxy-terminal amino acids of the GatA subunit only inhibits the Asp-AdT activity, leaving the Glu-AdT activity of the mutant AdT unaltered; this shows that Asp-AdT and Glu-AdT activities can be mechanistically separated.

Amino Acid Sequence↗
PubMed2000

Transfer RNA identity change in anticodon variants of E. coli tRNA(Phe) in vivo.

The anticodon sequence is a major recognition element for most aminoacyl-tRNA synthetases. We investigated the in vivo effects of changing the anticodon on the aminoacylation specificity in the example of E. coli tRNA(Phe). Constructing different anticodon mutants of E. coli tRNA(Phe) by site-directed mutagenesis, we isolated 22 anticodon mutant tRNA(Phe); the anticodons corresponded to 16 amino acids and an opal stop codon. To examine whether the mutant tRNAs had changed their amino acid acceptor specificity in vivo, we tested the viability of E. coli strains containing these tRNA(Phe) genes in a medium which permitted tRNA induction. Fourteen mutant tRNA genes did not affect host viability. However, eight mutant tRNA genes were toxic to the host and prevented growth, presumably because the anticodon mutants led to translational errors. Many mutant tRNAs which did not affect host viability were not aminoacylated in vivo. Three mutant tRNAs containing anticodon sequences corresponding to lysine (UUU), methionine (CAU) and threonine (UGU) were charged with the amino acid corresponding to their anticodon, but not with phenylalanine. These three tRNAs and tRNA(Phe) are located in the same cluster in a sequence similarity dendrogram of total E. coli tRNAs. The results support the idea that such tRNAs arising from in vivo evolution are derived by anticodon change from the same ancestor tRNA.

Anticodon↗
PubMed2000

One polypeptide with two aminoacyl-tRNA synthetase activities.

The genome sequences of certain archaea do not contain recognizable cysteinyl-transfer RNA (tRNA) synthetases, which are essential for messenger RNA-encoded protein synthesis. However, a single cysteinyl-tRNA synthetase activity was detected and purified from one such organism, Methanococcus jannaschii. The amino-terminal sequence of this protein corresponded to the predicted sequence of prolyl-tRNA synthetase. Biochemical and genetic analyses indicated that this archaeal form of prolyl-tRNA synthetase can synthesize both cysteinyl-tRNA(Cys) and prolyl-tRNA(Pro). The ability of one enzyme to provide two aminoacyl-tRNAs for protein synthesis raises questions about concepts of substrate specificity in protein synthesis and may provide insights into the evolutionary origins of this process.

Amino Acyl-tRNA Synthetases↗
PubMed2000

The adaptor hypothesis revisited.

As originally postulated in Crick's Adaptor hypothesis, the faithful synthesis of proteins from messenger RNA is dependent on the presence of perfectly acylated tRNAs. The hypothesis also suggested that each aminoacyl-tRNA would be made by a unique enzyme. Recent data have now forced a revision of this latter point, with an increasingly diverse array of enzymes and pathways being implicated in aminoacyl-tRNA synthesis. These unexpected findings have far-reaching implications for our understanding of protein synthesis and its origins.

Amino Acyl-tRNA Synthetases↗
PubMed2000

Antisense HEMA1 RNA expression inhibits heme and chlorophyll biosynthesis in arabidopsis.

5-aminolevulinic acid (ALA) is a precursor in the biosynthesis of tetrapyrroles including chlorophylls and heme. The formation of ALA involves two enzymatic steps which take place in the chloroplast in plants. The first enzyme, glutamyl-tRNA reductase, and the second enzyme, glutamate-1-semialdehyde-2,1-aminomutase, are encoded by the nuclear HEMA and GSA genes, respectively. To assess the significance of the HEMA gene for chlorophyll and heme synthesis, transgenic Arabidopsis plants that expressed antisense HEMA1 mRNA from the constitutive cauliflower mosaic virus 35S promoter were generated. These plants exhibited varying degrees of chlorophyll deficiency, ranging from patchy yellow to total yellow. Analysis indicated that these plants had decreased levels of chlorophyll, non-covalently bound hemes, and ALA; their levels were proportional to the level of glutamyl-tRNA reductase expression and were inversely related to the levels of antisense HEMA transcripts. Plants that lacked chlorophyll failed to survive under normal growth conditions, indicating that HEMA gene expression is essential for growth.

Aldehyde Oxidoreductases↗
PubMed2000

Cysteine biosynthesis pathway in the archaeon Methanosarcina barkeri encoded by acquired bacterial genes?

The pathway of cysteine biosynthesis in archaea is still unexplored. Complementation of a cysteine auxotrophic Escherichia coli strain NK3 led to the isolation of the Methanosarcina barkeri cysK gene [encoding O-acetylserine (thiol)-lyase-A], which displays great similarity to bacterial cysK genes. Adjacent to cysK is an open reading frame orthologous to bacterial cysE (serine transacetylase) genes. These two genes could account for cysteine biosynthesis in this archaeon. Analysis of recent genome data revealed the presence of bacteria-like cysM genes [encoding O-acetylserine (thiol)-lyase-B] in Pyrococcus spp., Sulfolobus solfataricus, and Thermoplasma acidophilum. However, no orthologs for these genes can be found in Methanococcus jannaschii, Methanobacterium thermoautotrophicum, and Archaeoglobus fulgidus, implying the existence of unrecognizable genes for the same function or a different cysteine biosynthesis pathway.

Acetyltransferases↗
PubMed2000

Aminoacyl-tRNA synthetases, the genetic code, and the evolutionary process.

The aminoacyl-tRNA synthetases (AARSs) and their relationship to the genetic code are examined from the evolutionary perspective. Despite a loose correlation between codon assignments and AARS evolutionary relationships, the code is far too highly structured to have been ordered merely through the evolutionary wanderings of these enzymes. Nevertheless, the AARSs are very informative about the evolutionary process. Examination of the phylogenetic trees for each of the AARSs reveals the following. (i) Their evolutionary relationships mostly conform to established organismal phylogeny: a strong distinction exists between bacterial- and archaeal-type AARSs. (ii) Although the evolutionary profiles of the individual AARSs might be expected to be similar in general respects, they are not. It is argued that these differences in profiles reflect the stages in the evolutionary process when the taxonomic distributions of the individual AARSs became fixed, not the nature of the individual enzymes. (iii) Horizontal transfer of AARS genes between Bacteria and Archaea is asymmetric: transfer of archaeal AARSs to the Bacteria is more prevalent than the reverse, which is seen only for the "gemini group. " (iv) The most far-ranging transfers of AARS genes have tended to occur in the distant evolutionary past, before or during formation of the primary organismal domains. These findings are also used to refine the theory that at the evolutionary stage represented by the root of the universal phylogenetic tree, cells were far more primitive than their modern counterparts and thus exchanged genetic material in far less restricted ways, in effect evolving in a communal sense.

Amino Acids↗
PubMed1999

Cysteinyl-tRNA formation: the last puzzle of aminoacyl-tRNA synthesis.

With the exception of the methanogenic archaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum deltaH, all organisms surveyed contain orthologs of Escherichia coli cysteinyl-tRNA synthetase (CysRS). The characterization of CysRS-encoding (cysS) genes and the demonstration of their ability to complement an E. coli cysSts mutant reveal that Methanococcus maripaludis and Methanosarcina barkeri, two other methanogenic archaea, possess canonical CysRS proteins. A molecular phylogeny inferred from 40 CysRS sequences indicates that the CysRS of M. maripaludis and Methanosarcina spp. are specific relatives of the CysRS of Pyrococcus spp. and Chlamydia, respectively. This result suggests that the CysRS gene was acquired by lateral gene transfer in at least one euryarchaeotic lineage.

Amino Acid Sequence↗
PubMed1999

Quality control mechanisms during translation.

Translation uses the genetic information in messenger RNA (mRNA) to synthesize proteins. Transfer RNAs (tRNAs) are charged with an amino acid and brought to the ribosome, where they are paired with the corresponding trinucleotide codon in mRNA. The amino acid is attached to the nascent polypeptide and the ribosome moves on to the next codon. The cycle is then repeated to produce a full-length protein. Proofreading and editing processes are used throughout protein synthesis to ensure the faithful translation of genetic information. The maturation of tRNAs and mRNAs is monitored, as is the identity of amino acids attached to tRNAs. Accuracy is further enhanced during the selection of aminoacyl-tRNAs on the ribosome and their base pairing with mRNA. Recent studies have begun to reveal the molecular mechanisms underpinning quality control and go some way to explaining the phenomenal accuracy of translation first observed over three decades ago.

Amino Acyl-tRNA Synthetases↗
PubMed1999

Transfer RNA identity contributes to transition state stabilization during aminoacyl-tRNA synthesis.

Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs ensure both accurate RNA recognition and the efficient catalysis of aminoacylation. The effects of tRNA(Trp)variants on the aminoacylation reaction catalyzed by wild-type Escherichia coli tryptophanyl-tRNA synthe-tase (TrpRS) have now been investigated by stopped-flow fluorimetry, which allowed a pre-steady-state analysis to be undertaken. This showed that tRNA(Trp)identity has some effect on the ability of tRNA to bind the reaction intermediate TrpRS-tryptophanyl-adenylate, but predominantly affects the rate at which trypto-phan is transferred from TrpRS-tryptophanyl adenylate to tRNA. Use of the binding ( K (tRNA)) and rate constants ( k (4)) to determine the energetic levels of the various species in the aminoacylation reaction showed a difference of approximately 2 kcal mol(-1)in the barrier to transition state formation compared to wild-type for both tRNA(Trp)A-->C73 and. These results directly show that tRNA identity contributes to the degree of complementarity to the transition state for tRNA charging in the active site of an aminoacyl-tRNA synthetase:aminoacyl-adenylate:tRNA complex.

Adenosine Monophosphate↗
PubMed1999

Substrate recognition by class I lysyl-tRNA synthetases: a molecular basis for gene displacement.

Lysyl-tRNA synthetases (LysRSs) are unique amongst the aminoacyl-tRNA synthetases in being composed of unrelated class I and class II enzymes. To allow direct comparison between the two types of LysRS, substrate recognition by class I LysRSs was examined. Genes encoding both an archaeal and a bacterial class I enzyme were able to rescue an Escherichia coli strain deficient in LysRS, indicating their ability to functionally substitute for a class II LysRS in vivo. In vitro characterization showed lysine activation and recognition to be tRNA-dependent, an attribute of several class I, but not class II, aminoacyl-tRNA synthetases. Examination of tRNA recognition showed that class I LysRSs recognize the same elements in tRNALys as their class II counterparts, namely the discriminator base (N73) and the anticodon. This sequence-specific recognition of the same nucleotides in tRNALys by the two unrelated types of enzyme suggests that tRNALys predates at least one of the LysRSs in the evolution of the translational apparatus. The only observed variation in recognition was that the G2.U71 wobble pair of spirochete tRNALys acts as antideterminant for class II LysRS but does not alter class I enzyme recognition. This difference in tRNA recognition strongly favors the use of a class I-type enzyme to aminoacylate particular tRNALys species and provides a molecular basis for the observed displacement of class II by class I LysRSs in certain bacteria.

Amino Acyl-tRNA Synthetases↗
PubMed1999

Mutations in a new Arabidopsis cyclophilin disrupt its interaction with protein phosphatase 2A.

The heterotrimeric protein phosphatase 2A (PP2A) is a component of multiple signaling pathways in eukaryotes. Disruption of PP2A activity in Arabidopsis is known to alter auxin transport and growth response pathways. We demonstrated that the regulatory subunit A of an Arabidopsis PP2A interacts with a novel cyclophilin, ROC7. The gene for this cyclophilin encodes a protein that contains a unique 30-amino acid extension at the N-terminus, which distinguishes the gene product from all previously identified Arabidopsis cyclophilins. Altered forms of ROC7 cyclophilin with mutations in the conserved DENFKL domain did not bind to PP2A. Unlike protein phosphatase 2B, PP2A activity in Arabidopsis extracts was not affected by the presence of the cyclophilin-binding molecule cyclosporin. The ROC7 transcript was expressed to high levels in all tissues tested. Expression of an ROC7 antisense transcript gave rise to increased root growth. These results indicate that cyclophilin may have a role in regulating PP2A activity, by a mechanism that differs from that employed for cyclophilin regulation of PP2B.

Amino Acid Sequence↗
PubMed1999

Selective inhibition of HEMA gene expression by photooxidation in Arabidopsis thaliana.

Norflurazon (NF), a photobleaching herbicide, inhibits carotenoid biosynthesis. Lack of carotenoid pigments leads to photooxidative damage of chloroplasts. In this study of Arabidopsis thaliana we demonstrate that NF-treated photobleached plants are still able to make 5-aminolevulinic acid (ALA) the first precursor of porphyrins and tetrapyrroles. ALA is formed in the tRNA-dependent two-step C5-pathway in the chloroplast of plants. The expression of glutamyl-tRNA reductase (GluTR), the first enzyme in the pathway, was severely inhibited by NF, while treatment with this compound did not significantly reduce the levels of the other enzyme, glutamate-l-semialdehyde aminomutase, or of tRNA(Glu), the initial metabolite of the pathway. Extracts of these plants retained the capacity, albeit reduced, to convert exogenously added glutamate to ALA. Thus, the much-reduced level of ALA formation in photobleached plants is due to selective inhibition of GluTR expression.

Aldehyde Oxidoreductases↗
PubMed1999

The RCN1-encoded A subunit of protein phosphatase 2A increases phosphatase activity in vivo.

Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic C subunit and two distinct regulatory subunits, A and B. The RCN1 gene encodes one of three A regulatory subunits in Arabidopsis thaliana. A T-DNA insertion mutation at this locus impairs root curling, seedling organ elongation and apical hypocotyl hook formation. We have used in vivo and in vitro assays to gauge the impact of the rcn1 mutation on PP2A activity in seedlings. PP2A activity is decreased in extracts from rcn1 mutant seedlings, and this decrease is not due to a reduction in catalytic subunit expression. Roots of mutant seedlings exhibit increased sensitivity to the phosphatase inhibitors okadaic acid and cantharidin in organ elongation assays. Shoots of dark-grown, but not light-grown seedlings also show increased inhibitor sensitivity. Furthermore, cantharidin treatment of wild-type seedlings mimics the rcn1 defect in root curling, root waving and hypocotyl hook formation assays. In roots of wild-type seedlings, RCN1 mRNA is expressed at high levels in root tips, and accumulates to lower levels in the pericycle and lateral root primordia. In shoots, RCN1 is expressed in the apical hook and the basal, rapidly elongating cells in etiolated hypocotyls, and in the shoot meristem and leaf primordia of light-grown seedlings. Our results show that the wild-type RCN1-encoded A subunit functions as a positive regulator of the PP2A holoenzyme, increasing activity towards substrates involved in organ elongation and differential cell elongation responses such as root curling.

Arabidopsis↗
PubMed1999

Archaeal aminoacyl-tRNA synthesis: diversity replaces dogma.

Accurate aminoacyl-tRNA synthesis is essential for faithful translation of the genetic code and consequently has been intensively studied for over three decades. Until recently, the study of aminoacyl-tRNA synthesis in archaea had received little attention. However, as in so many areas of molecular biology, the advent of archaeal genome sequencing has now drawn researchers to this field. Investigations with archaea have already led to the discovery of novel pathways and enzymes for the synthesis of numerous aminoacyl-tRNAs. The most surprising of these findings has been a transamidation pathway for the synthesis of asparaginyl-tRNA and a novel lysyl-tRNA synthetase. In addition, seryl- and phenylalanyl-tRNA synthetases that are only marginally related to known examples outside the archaea have been characterized, and the mechanism of cysteinyl-tRNA formation in Methanococcus jannaschii and Methanobacterium thermoautotrophicum is still unknown. These results have revealed completely unexpected levels of complexity and diversity, questioning the notion that aminoacyl-tRNA synthesis is one of the most conserved functions in gene expression. It has now become clear that the distribution of the various mechanisms of aminoacyl-tRNA synthesis in extant organisms has been determined by numerous gene transfer events, indicating that, while the process of protein biosynthesis is orthologous, its constituents are not.

Amino Acyl-tRNA Synthetases↗
PubMed1998

C-terminal truncation of yeast SerRS is toxic for Saccharomyces cerevisiae due to altered mechanism of substrate recognition.

Like all other eukaryal cytosolic seryl-tRNA synthetase (SerRS) enzymes, Saccharomyces cerevisiae SerRS contains a C-terminal extension not found in the enzymes of eubacterial and archaeal origin. Overexpression of C-terminally truncated SerRS lacking the 20-amino acid appended domain (SerRSC20) is toxic to S. cerevisiae possibly because of altered substrate recognition. Compared to wild-type SerRS the truncated enzyme displays impaired tRNA-dependent serine recognition and is less stable. This suggests that the C-terminal peptide is important for the formation or maintenance of the enzyme structure optimal for substrate binding and catalysis.

Amino Acid Sequence↗