Biomedical subjects

Dieter Söll

Publications and source records attributed to Dieter Söll.

16 recordsLinked to original sources

PubMed2003

The structural basis of cysteine aminoacylation of tRNAPro by prolyl-tRNA synthetases.

Cysteinyl-tRNA synthetase is an essential enzyme required for protein synthesis. Genes encoding this protein have not been identified in Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, or Methanopyrus kandleri. It has previously been proposed that the prolyl-tRNA synthetase (ProRS) enzymes in these organisms recognize either proline or cysteine and can aminoacylate their cognate tRNAs through a dual-specificity mechanism. We report five crystal structures at resolutions between 2.6 and 3.2 A: apo M. jannaschii ProRS, and M. thermautotrophicus ProRS in apo form and in complex with cysteinyl-sulfamoyl-, prolyl-sulfamoyl-, and alanyl-sulfamoyl-adenylates. These aminoacyl-adenylate analogues bind to a single active-site pocket and induce an identical set of conformational changes in loops around the active site when compared with the ligand-free conformation of ProRS. The cysteinyl- and prolyl-adenylate analogues have similar, nanomolar affinities for M. thermautotrophicus ProRS. Homology modeling of tRNA onto these adenylate complexes places the 3'-OH of A76 in an appropriate position for the transfer of any of the three amino acids to tRNA. Thus, these structures explain recent biochemical experiments showing that M. jannaschii ProRS misacylates tRNA(Pro) with cysteine, and argue against the proposal that these archaeal ProRS enzymes possess the dual capacity to aminoacylate both tRNA(Pro) and tRNA(Cys) with their cognate amino acids.

Acylation↗

PubMed2003

Protein synthesis in Escherichia coli with mischarged tRNA.

Two types of aspartyl-tRNA synthetase exist: the discriminating enzyme (D-AspRS) forms only Asp-tRNA(Asp), while the nondiscriminating one (ND-AspRS) also synthesizes Asp-tRNA(Asn), a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). On the basis of the E. coli trpA34 missense mutant transformed with heterologous ND-aspS genes, we developed a system with which to measure the in vivo formation of Asp-tRNA(Asn) and its acceptance by elongation factor EF-Tu. While large amounts of Asp-tRNA(Asn) are detrimental to E. coli, smaller amounts support protein synthesis and allow the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase.

Aspartate-tRNA Ligase↗

PubMed2002

Orthologs of a novel archaeal and of the bacterial peptidyl-tRNA hydrolase are nonessential in yeast.

Peptidyl-tRNA hydrolase (encoded by pth) is an essential enzyme in all bacteria, where it releases tRNA from the premature translation termination product peptidyl-tRNA. Archaeal genomes lack a recognizable peptidyl-tRNA hydrolase (Pth) ortholog, although it is present in most eukaryotes. However, we detected Pth-like activity in extracts of the archaeon Methanocaldococcus jannaschii. The uncharacterized MJ0051 ORF was shown to correspond to a protein with Pth activity. Heterologously expressed MJ0051 enzyme catalyzed in vitro the cleavage of the Pth substrates diacetyl-[14C]lysyl-tRNA and acetyl-[14C]phenylalanyl-tRNA. On transformation of an Escherichia coli pth(ts) mutant, the MJ0051 gene (named pth2) rescued the temperature-sensitive phenotype of the strain. Analysis of known genomes revealed the presence of highly conserved orthologs of the archaeal pth2 gene in all archaea and eukaryotes but not in bacteria. The phylogeny of pth2 homologs suggests that the gene has been vertically inherited throughout the archaeal and eukaryal domains. Deletions in Saccharomyces cerevisiae of the pth2 (YBL057c) or pth (YHR189w) orthologs were viable, as was the double deletion strain, implying that the canonical Pth and Pth2 enzymes are not essential for yeast viability.

Amino Acid Sequence↗

PubMed2002

A one-step method for in vitro production of tRNA transcripts.

Sequencing of a large number of microbial genomes has led to the discovery of new enzymes involved in tRNA biosynthesis and tRNA function. Preparation of a great variety of RNA molecules is, therefore, of major interest for biochemical characterization of these proteins. We describe a fast, cost-effective and efficient method for in vitro production of tRNA transcripts. T7 RNA polymerase requires a double-stranded DNA promoter in order to initiate transcription; however, elongation does not require a double-stranded DNA template. A partially double-stranded transcription template formed by annealing of a short oligonucleotide, complementary to the T7 promoter, to a larger oligonucleotide is shown to be a good substrate for in vitro transcription. This method allows rapid production of a variety of tRNA transcripts which can be aminoacylated well. This eliminates the need for cloning of tRNA genes, large-scale plasmid preparation and enzymatic digestion.

DNA↗

PubMed2002

Escherichia coli glutamyl-tRNA reductase. Trapping the thioester intermediate.

In the first step of tetrapyrrole biosynthesis in Escherichia coli, glutamyl-tRNA reductase (GluTR, encoded by hemA) catalyzes the NADPH-dependent reduction of glutamyl-tRNA to glutamate-1-semialdehyde. Soluble homodimeric E. coli GluTR was made by co-expressing the hemA gene and the chaperone genes dnaJK and grpE. During Mg(2+)-stimulated catalysis, the reactive sulfhydryl group of Cys-50 in the E. coli enzyme attacks the alpha-carbonyl group of the tRNA-bound glutamate. The resulting thioester intermediate was trapped and detected by autoradiography. In the presence of NADPH, the end product, glutamate-1-semialdehyde, is formed. In the absence of NADPH, E. coli GluTR exhibited substrate esterase activity. The in vitro synthesized unmodified glutamyl-tRNA was an acceptable substrate for E. coli GluTR. Eight 5-aminolevulinic acid auxotrophic E. coli hemA mutants were genetically selected, and the corresponding mutations were determined. Most of the recombinant purified mutant GluTR enzymes lacked detectable activity. Based on the Methanopyrus kandleri GluTR structure, the positions of the amino acid exchanges are close to the catalytic domain (G7D, E114K, R314C, S22L/S164F, G44C/S105N/A326T, G106N, S145F). Only GluTR G191D (affected in NADPH binding) revealed esterase but no reductase activity.

Aldehyde Oxidoreductases↗

PubMed2002

Evolutionary divergence of the archaeal aspartyl-tRNA synthetases into discriminating and nondiscriminating forms.

Asparaginyl-tRNA (Asn-tRNA) is generated in nature via two alternate routes, either direct acylation of tRNA with asparagine by asparaginyl-tRNA synthetase (AsnRS) or in a two-step pathway that requires misacylated Asp-tRNA(Asn) as an intermediate. This misacylated aminoacyl-tRNA is formed by a nondiscriminating aspartyl-tRNA synthetase (AspRS), an enzyme that in addition to forming Asp-tRNA(Asp) also misacylates tRNA(Asn). In contrast, a discriminating AspRS cannot acylate tRNA(Asn). It has been suggested that the archaeal AspRS enzymes are nondiscriminating, whereas the bacterial ones discriminate. The archaeal and bacterial AspRS proteins are indeed distinct in sequence and structure. However, we show that both discriminating and nondiscriminating forms of AspRS exist among the archaea. Using unfractionated methanobacterial and pyrococcal tRNA, the Methanothermobacter thermautotrophicus AspRS acylated approximately twice as much tRNA as did AspRS from Pyrococcus kodakaraensis or Ferroplasma acidarmanus. Proof that Asp-tRNA(Asn) was generated by the methanogen synthetase was the conversion of Asp-tRNA formed by M. thermautotrophicus AspRS to Asn-tRNA by M. thermautotrophicus Asp-tRNA(Asn) amidotransferase. In contrast, Asp-tRNA formed by the Pyrococcus or Ferroplasma enzymes was not a substrate for the amidotransferase. Also, although all three AspRS enzymes charged tRNA(Asp) transcripts, only M. thermautotrophicus AspRS aspartylated the tRNA(Asn) transcript. Genomic analysis provides a rationale for the nature of these enzymes. The mischarging AspRS correlates with the absence in the genome of AsnRS and the presence of Asp-tRNA(Asn) amidotransferase, employed by the transamidation pathway. In contrast, the discriminating AspRS correlates with the absence of the amidotransferase and the presence of AsnRS, forming Asn-tRNA by direct aminoacylation. The high sequence identity, up to 60% between discriminating and nondiscriminating archaeal AspRSs, suggests that few mutational steps may be necessary to convert the tRNA-discriminating ability of a tRNA synthetase.

Aspartate-tRNA Ligase↗

PubMed2002

Cysteine activation is an inherent in vitro property of prolyl-tRNA synthetases.

Aminoacyl-tRNA synthetases are well known for their remarkable precision in substrate selection during aminoacyl-tRNA formation. Some synthetases enhance the accuracy of this process by editing mechanisms that lead to hydrolysis of incorrectly activated and/or charged amino acids. Prolyl-tRNA synthetases (ProRSs) can be divided into two structurally divergent groups, archaeal-type and bacterial-type enzymes. A striking difference between these groups is the presence of an insertion domain (approximately 180 amino acids) in the bacterial-type ProRS. Because the archaeal-type ProRS enzymes have been shown to recognize cysteine, we tested selected ProRSs from all three domains of life to determine whether cysteine activation is a general property of ProRS. Here we show that cysteine is activated by recombinant ProRS enzymes from the archaea Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus, from the eukaryote Saccharomyces cerevisiae, and from the bacteria Aquifex aeolicus, Borrelia burgdorferi, Clostridium sticklandii, Cytophaga hutchinsonii, Deinococcus radiodurans, Escherichia coli, Magnetospirillum magnetotacticum, Novosphingobium aromaticivorans, Rhodopseudomonas palustris, and Thermus thermophilus. This non-cognate amino acid was efficiently acylated in vitro onto tRNA(Pro), and the misacylated Cys-tRNA(Pro) was not edited by ProRS. Therefore, ProRS exhibits a natural level of mischarging that is to date unequalled among the aminoacyl-tRNA synthetases.

Amino Acid Sequence↗

PubMed2002

Methanocaldococcus jannaschii prolyl-tRNA synthetase charges tRNA(Pro) with cysteine.

Methanocaldococcus jannaschii prolyl-tRNA synthetase (ProRS) was previously reported to also catalyze the synthesis of cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) to make up for the absence of the canonical cysteinyl-tRNA synthetase in this organism (Stathopoulos, C., Li, T., Longman, R., Vothknecht, U. C., Becker, H., Ibba, M., and Söll, D. (2000) Science 287, 479-482; Lipman, R. S., Sowers, K. R., and Hou, Y. M. (2000) Biochemistry 39, 7792-7798). Here we show by acid urea gel electrophoresis that pure heterologously expressed recombinant M. jannaschii ProRS misaminoacylates M. jannaschii tRNA(Pro) with cysteine. The enzyme is unable to aminoacylate purified mature M. jannaschii tRNA(Cys) with cysteine in contrast to facile aminoacylation of the same tRNA with cysteine by Methanococcus maripaludis cysteinyl-tRNA synthetase. Although M. jannaschii ProRS catalyzes the synthesis of Cys-tRNA(Pro) readily, the enzyme is unable to edit this misaminoacylated tRNA. We discuss the implications of these results on the in vivo activity of the M. jannaschii ProRS and on the nature of the enzyme involved in the synthesis of Cys-tRNA(Cys) in M. jannaschii.

Acylation↗

PubMed2002

Genetic code: introducing pyrrolysine.

Monomethylamine methyltransferase of the archaebacterium Methanosarcina barkeri contains a novel amino acid, pyrrolysine, encoded by the termination codon UAG. Initial studies suggest that pyrrolysine may be co-translationally inserted during protein synthesis, probably by a mechanism analogous to that operating during selenocysteine incorporation.

Amino Acyl-tRNA Synthetases↗

PubMed2002

Indolmycin resistance of Streptomyces coelicolor A3(2) by induced expression of one of its two tryptophanyl-tRNA synthetases.

Aminoacyl-tRNA synthetases, a family of enzymes essential for protein synthesis, are promising targets of antimicrobials. Indolmycin, a secondary metabolite of Streptomyces griseus and a selective inhibitor of prokaryotic tryptophanyl-tRNA synthetase (TrpRS), was used to explore the mechanism of inhibition and to explain the resistance of a naturally occurring strain. Streptomyces coelicolor A3(2), an indolmycin-resistant strain, contains two trpS genes encoding distinct TrpRS enzymes. We show that TrpRS1 is indolmycin-resistant in vitro and in vivo, whereas TrpRS2 is sensitive. The lysine (position 9) in the enzyme tryptophan binding site is essential for making TrpRS1 indolmycin-resistant. Replacement of lysine 9 by glutamine, which at this position is conserved in most bacterial TrpRS proteins, abolished the ability of the mutant trpS gene to confer indolmycin resistance in vivo. Molecular modeling suggests that lysine 9 sterically hinders indolmycin binding to the enzyme. Tryptophan recognition (assessed by k(cat)/K(M)) by TrpRS1 is 4-fold lower than that of TrpRS2. Examination of the mRNA for the two enzymes revealed that only TrpRS2 mRNA is constitutively expressed, whereas mRNA for the indolmycin-resistant TrpRS1 enzyme is induced when the cells are exposed to indolmycin.

Amino Acid Sequence↗

PubMed2002

Cysteinyl-tRNA formation and prolyl-tRNA synthetase.

Aminoacyl-tRNA (AA-tRNA) formation is a key step in protein biosynthesis. This reaction is catalyzed with remarkable accuracy by the AA-tRNA synthetases, a family of 20 evolutionarily conserved enzymes. The lack of cysteinyl-tRNA (Cys-tRNA) synthetase in some archaea gave rise to the discovery of the archaeal prolyl-tRNA (Pro-tRNA) synthetase, an enzyme capable of synthesizing Pro-tRNA and Cys-tRNA. Here we review our current knowledge of this fascinating process.

Amino Acyl-tRNA Synthetases↗

PubMed2002

Transfer RNA-dependent amino acid biosynthesis: an essential route to asparagine formation.

Biochemical experiments and genomic sequence analysis showed that Deinococcus radiodurans and Thermus thermophilus do not possess asparagine synthetase (encoded by asnA or asnB), the enzyme forming asparagine from aspartate. Instead these organisms derive asparagine from asparaginyl-tRNA, which is made from aspartate in the tRNA-dependent transamidation pathway [Becker, H. D. & Kern, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12832-12837; and Curnow, A. W., Tumbula, D. L., Pelaschier, J. T., Min, B. & Söll, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12838-12843]. A genetic knockout disrupting this pathway deprives D. radiodurans of the ability to synthesize asparagine and confers asparagine auxotrophy. The organism's capacity to make asparagine could be restored by transformation with Escherichia coli asnB. This result demonstrates that in Deinococcus, the only route to asparagine is via asparaginyl-tRNA. Analysis of the completed genomes of many bacteria reveal that, barring the existence of an unknown pathway of asparagine biosynthesis, a wide spectrum of bacteria rely on the tRNA-dependent transamidation pathway as the sole route to asparagine.

Amino Acids↗

PubMed2002

Aminoacyl-tRNA formation in the extreme thermophile Thermus thermophilus.

Thermophilic organisms must be capable of accurate translation at temperatures in which the individual components of the translation machinery and also specific amino acids are particularly sensitive. Thermus thermophilus is a good model organism for studies of thermophilic translation because many of the components in this process have undergone structural and biochemical characterization. We have focused on the pathways of aminoacyl-tRNA synthesis for glutamine, asparagine, proline, and cysteine. We show that the T. thermophilus prolyl-tRNA synthetase (ProRS) exhibits cysteinyl-tRNA synthetase (CysRS) activity although the organism also encodes a canonical CysRS. The ProRS requires tRNA for cysteine activation, as is known for the characterized archaeal prolyl-cysteinyl-tRNA synthetase (ProCysRS) enzymes. The heterotrimeric T. thermophilus aspartyl-tRNA(Asn) amidotransferase can form Gln-tRNA in addition to Asn-tRNA: however, a 13-amino-acid C-terminal truncation of the holoenzyme A subunit is deficient in both activities when assayed with homologous substrates. A survey of codon usage in completed prokaryotic genomes identified a higher Glu:Gln ratio in proteins of thermophiles compared to mesophiles.

Amino Acyl-tRNA Synthetases↗

PubMed2002

Divergent regulation of the HEMA gene family encoding glutamyl-tRNA reductase in Arabidopsis thaliana: expression of HEMA2 is regulated by sugars, but is independent of light and plastid signalling.

The synthesis of 5-aminolevulinic acid (ALA) is a key regulatory step for the production of hemes and chlorophyll via the tetrapyrrole synthesis pathway. The first enzyme committed to ALA synthesis is glutamyl-tRNA reductase encoded in Arabidopsis by a small family of nuclear-encoded HEMA genes. To better understand the regulation of the tetrapyrrole synthesis pathway we have made a detailed study of HEMA2 expression with transgenic Arabidopsis thaliana L. Col. plants carrying chimeric HEMA2 promoter:gusA fusion constructs. Our results show that the HEMA2 promoter directs expression predominantly to roots and flowers, but that HEMA2 is also expressed at low levels in photosynthetic tissues. Deletion analysis of the HEMA2 promoter indicates that a ca. 850 bp fragment immediately upstream of the HEMA2 coding region is sufficient to drive regulated gusA expression. In contrast to HEMA1, HEMA2 is not up-regulated by red, far-red, blue, UV or white light. In addition, elimination of a promotive plastid signal by Norflurazon-induced photobleaching of plastids had no effect on HEMA2 expression while being required for normal white-light induction of HEMA1. HEMA2 expression in the cotyledons is inhibited by the presence of sucrose or glucose, but not fructose, and this response is light-independent. HEMA1 expression in cotyledons is also inhibited by sugars, but in a strictly light-dependent manner. The roles of HEMA1 and HEMA2 in meeting cellular tetrapyrrole requirements are discussed.

Aldehyde Oxidoreductases↗

PubMed2002

Functional convergence of two lysyl-tRNA synthetases with unrelated topologies.

Lysyl-tRNA can be synthesized by both a class I (LysRS-I) and a class II (LysRS-II) lysyl-tRNA synthetase. The crystal structure of LysRS-I from Pyrococcus horikoshii at 2.6 A resolution reveals extensive similarity with glutamyl-tRNA synthetase (GluRS). A comparison of the structures of LysRS-I and LysRS-II in complex with lysine shows that both enzymes use similar strategies for substrate recognition within unrelated active site topologies. A docking model based upon the GluRS-tRNA complex suggests how LysRS-I and LysRS-II can recognize the same molecular determinants in tRNALys, as shown by biochemical results, while approaching the acceptor helix of the tRNA from opposite sides.

Acylation↗

PubMed2002

tRNA-dependent amino acid discrimination by yeast seryl-tRNA synthetase.

The ability of aminoacyl-tRNA synthetases to distinguish between similar amino acids is crucial for accurate translation of the genetic code. Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) employs tRNA-dependent recognition of its cognate amino acid serine [Lenhard, B., Filipic, S., Landeka, I., Skrtic, I., Söll, D. & Weygand-Durasevic, I. (1997) J. Biol. Chem.272, 1136-1141]. Here we show that dimeric SerRS enzyme complexed with one molecule of tRNASer is more specific and more efficient in catalyzing seryl-adenylate formation than the apoenzyme alone. Sequence-specific tRNA-protein interactions enhance discrimination of the amino acid substrate by yeast SerRS and diminish the misactivation of the structurally similar noncognate threonine. This may proceed via a tRNA-induced conformational change in the enzyme's active site. The 3'-terminal adenosine of tRNASer is not important in effecting the rearrangement of the serine binding site. Our results do not provide an indication for a readjustment of ATP binding in a tRNA-assisted manner. The stoichiometric analyses of the complexes between the enzyme and tRNASer revealed that two cognate tRNA molecules can be bound to dimeric SerRS, however, with very different affinities.

Adenosine Triphosphate↗