A snapshot of the Physcomitrella N-terminome reveals N-terminal methylation of organellar proteins.

Analysis of the N-terminome of Physcomitrella reveals N-terminal monomethylation of nuclear-encoded, mitochondria-localized proteins. Post- or co-translational N-terminal modifications of proteins influence their half-life as well as mediating protein sorting to organelles via cleavable N-terminal sequences that are recognized by the respective translocation machinery. Here, we provide an overview on the current modification state of the N-termini of over 4500 proteins from the model moss Physcomitrella (Physcomitrium patens) using a compilation of 24 N-terminomics datasets.

Elucidating the structure-function attributes of a trypanosomal arginyl-tRNA synthetase.

Aminoacyl-tRNA synthetases (aaRSs) are fundamental components of the protein translation machinery. In light of their pivotal role in protein synthesis and structural divergence among species, they have always been considered potential targets for the development of antimicrobial compounds. Arginyl-tRNA synthetase from Trypanosoma cruzi (TcArgRS), the parasite responsible for causing Chagas Disease, contains a 100-amino acid insertion that was found to be completely absent in the human counterpart of similar length, as ascertained from multiple sequence alignment results.

Evolutionary Adjustment of tRNA Identity Rules in Bacillariophyta for Recognition by an Aminoacyl-tRNA Synthetase Adds a Facet to the Origin of Diatoms.

Error-free protein synthesis relies on the precise recognition by the aminoacyl-tRNA synthetases of their cognate tRNAs in order to attach the corresponding amino acid. A concept of universal tRNA identity elements requires the aminoacyl-tRNA synthetases provided by the genome of an organism to match the identity elements found in the cognate tRNAs in an evolution-independent manner. Identity elements tend to cluster in the tRNA anticodon and acceptor stem regions.

The Evolutionary Fate of Mitochondrial Aminoacyl-tRNA Synthetases in Amitochondrial Organisms.

During the endosymbiotic evolution of mitochondria, the genes for aminoacyl-tRNA synthetases were transferred to the ancestral nucleus. A further reduction of mitochondrial function resulted in mitochondrion-related organisms (MRO) with a loss of the organelle genome. The fate of the now redundant ancestral mitochondrial aminoacyl-tRNA synthetase genes is uncertain.

Molecular evidence for the evolution of the eukaryotic mitochondrial arginyl-tRNA synthetase from the prokaryotic suborder Cystobacterineae.

The evolutionary origin of the family of eukaryotic aminoacyl-tRNA synthetases that are essential to all living organisms is a matter of debate. In order to shed molecular light on the ancient source of arginyl-tRNA synthetase, a total of 1347 eukaryotic arginyl-tRNA synthetase sequences were mined from databases and analyzed. Their multiple sequence alignment reveals a signature sequence that is characteristic of the nuclear-encoded enzyme, which is imported into mitochondria.

Identification of Targets and Interaction Partners of Arginyl-tRNA Protein Transferase in the Moss Physcomitrella patens.

Protein arginylation is a posttranslational modification of both N-terminal amino acids of proteins and sidechain carboxylates and can be crucial for viability and physiology in higher eukaryotes. The lack of arginylation causes severe developmental defects in moss, affects the low oxygen response in Arabidopsis thaliana and is embryo lethal in Drosophila and in mice. Although several studies investigated impact and function of the responsible enzyme, the arginyl-tRNA protein transferase (ATE) in plants, identification of arginylated proteins by mass spectrometry was not hitherto achieved.

Where have all the inosines gone? Conflicting evidence for A-to-I editing of the anticodon of higher eukaryotic tRNAACGArg questions the dogma of a universal wobble-mediated decoding of CGN codons.

Codon-anticodon recognition between triplets of an mRNA and a specific tRNA is the key element in the translation of the genetic code. In general, the precision of this process is dominated by a strict Watson-Crick base-pairing scheme. However, the degeneracy of the genetic code led Crick to propose the Wobble Hypothesis, permitting a less restraining interaction with the third base of the codon and involving the participation of inosine for decoding C-ending codons.

Spatio-temporal patterning of arginyl-tRNA protein transferase (ATE) contributes to gametophytic development in a moss.

The importance of the arginyl-tRNA protein transferase (ATE), the enzyme mediating post-translation arginylation of proteins in the N-end rule degradation (NERD) pathway of protein stability, was analysed in Physcomitrella patens and compared to its known functions in other eukaryotes. We characterize ATE:GUS reporter lines as well as ATE mutants in P. patens to study the impact and function of arginylation on moss development and physiology.

Identity elements for the aminoacylation of metazoan mitochondrial tRNA(Arg) have been widely conserved throughout evolution and ensure the fidelity of the AGR codon reassignment.

Eumetazoan mitochondrial tRNAs possess structures (identity elements) that require the specific recognition by their cognate nuclear-encoded aminoacyl-tRNA synthetases. The AGA (arginine) codon of the standard genetic code has been reassigned to serine/glycine/termination in eumetazoan organelles and is translated in some organisms by a mitochondrially encoded tRNA(Ser)UCU. One mechanism to prevent mistranslation of the AGA codon as arginine would require a set of tRNA identity elements distinct from those possessed by the cytoplasmic tRNAArg in which the major identity elements permit the arginylation of all 5 encoded isoacceptors.

Amino acid discrimination by the nuclear encoded mitochondrial arginyl-tRNA synthetase of the larva of a bruchid beetle (Caryedes brasiliensis) from northwestern Costa Rica.

L-canavanine, the toxic guanidinooxy analogue of L-arginine, is the product of plant secondary metabolism. The need for a detoxifying mechanism for the producer plant is self-evident but the larvae of the bruchid beetle Caryedes brasiliensis, that is itself a non-producer, have specialized in feeding on the Lcanavanine-containing seeds of Dioclea megacarpa. The evolution of a seed predator that can imitate the enzymatic abilities of the host permits us to address the question of whether the same problem of amino acid recognition in two different kingdoms has been solved by the same mechanism.

Small RNA library construction from minute biological samples.

Increasingly, the discovery and characterization of small regulatory RNAs from a variety of organisms have all required deep-sequencing methodologies. However, the crux to successful deep-sequencing analysis depends upon optimal construction of a cDNA library compatible for the high-throughput sequencing platform. Challenges to small RNA library constructions arise when dealing with minute tissue samples because certain structural RNA fragments can dominate and mask the desired characterization of regulatory small RNAs like microRNAs (miRNAs), endogenous small interfering RNAs (endo-siRNAs), and Piwi-interacting RNAs (piRNAs).

The absence of A-to-I editing in the anticodon of plant cytoplasmic tRNA (Arg) ACG demands a relaxation of the wobble decoding rules.

It is a prevalent concept that, in line with the Wobble Hypothesis, those tRNAs having an adenosine in the first position of the anticodon become modified to an inosine at this position. Sequencing the cDNA derived from the gene coding for cytoplasmic tRNA (Arg) ACG from several higher plants as well as mass spectrometric analysis of the isoacceptor has revealed that for this kingdom an unmodified A in the wobble position of the anticodon is the rule rather than the exception. In vitro translation shows that in the plant system the absence of inosine in the wobble position of tRNA (Arg) does not prevent decoding.

The influence of identity elements on the aminoacylation of tRNA(Arg) by plant and Escherichia coli arginyl-tRNA synthetases.

Identity elements determine the accurate recognition between tRNAs and aminoacyl-tRNA synthetases. The arginine system from yeast and Escherichia coli has been studied extensively in the past. However, information about the enzymes from higher eukaryotes is limited and plant aminoacyl-tRNA synthetases have been largely ignored in this respect.

Amino acid discrimination by arginyl-tRNA synthetases as revealed by an examination of natural specificity variants.

L-canavanine occurs as a toxic non-protein amino acid in more than 1500 leguminous plants. One mechanism of its toxicity is its incorporation into proteins, replacing L-arginine and giving rise to functionally aberrant polypeptides. A comparison between the recombinant arginyl-tRNA synthetases from a canavanine producer (jack bean) and from a related non-producer (soybean) provided an opportunity to study the mechanism that has evolved to discriminate successfully between the proteinogenic amino acid and its non-protein analogue.

Expression and properties of arginyl-tRNA synthetase from jack bean (Canavalia ensiformis).

The coding region for arginyl-tRNA synthetase from jack bean (Canavalia ensiformis) has been sequenced and cloned into the bacterial expression vector pET32a. Transformation of BL21 cells and induction with IPTG results in the high level expression of the protein fused N-terminally with thioredoxin and bearing a His-tag. A substantial proportion of the enzyme is recovered in the soluble fraction of the cell lysate (10 mg per litre cell culture) and can be isolated with metal-affinity technology.

Two closely related pathways of nicotine catabolism in Arthrobacter nicotinovorans and Nocardioides sp. strain JS614.

A virtually identical nicotine catabolic pathway including the heterotrimeric molybdenum enzyme nicotine and 6-hydroxy-pseudo-oxynicotine dehydrogenase, 6-hydroxy-L: -nicotine oxidase, 2,6-dihydroxy-pseudo-oxynicotine hydrolase, and 2,6-dihydroxypyridine hydroxylase have been identified in A. nicotinovorans and Nocardioides sp. JS614.

A two-component small multidrug resistance pump functions as a metabolic valve during nicotine catabolism by Arthrobacter nicotinovorans.

The genes nepAB of a small multidrug resistance (SMR) pump were identified as part of the pAO1-encoded nicotine regulon responsible for nicotine catabolism in Arthrobacter nicotinovorans. When [(14)C]nicotine was added to the growth medium the bacteria exported the (14)C-labelled end product of nicotine catabolism, methylamine. In the presence of the proton-motive force inhibitors 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenylhydrazone (CCCP) or the proton ionophore nigericin, export of methylamine was inhibited and radioactivity accumulated inside the bacteria.

Final steps in the catabolism of nicotine.

New enzymes of nicotine catabolism instrumental in the detoxification of the tobacco alkaloid by Arthrobacter nicotinovorans pAO1 have been identified and characterized. Nicotine breakdown leads to the formation of nicotine blue from the hydroxylated pyridine ring and of gamma-N-methylaminobutyrate (CH(3)-4-aminobutyrate) from the pyrrolidine ring of the molecule. Surprisingly, two alternative pathways for the final steps in the catabolism of CH(3)-4-aminobutyrate could be identified.

An alpha/beta-fold C--C bond hydrolase is involved in a central step of nicotine catabolism by Arthrobacter nicotinovorans.

The enzyme catalyzing the hydrolytic cleavage of 2,6-dihydroxypseudooxynicotine to 2,6-dihydroxypyridine and gamma-N-methylaminobutyrate was found to be encoded on pAO1 of Arthrobacter nicotinovorans. The new enzyme answers an old question about nicotine catabolism and may be the first C--C bond hydrolase that is involved in the biodegradation of a heterocyclic compound.

Sequence of the 165-kilobase catabolic plasmid pAO1 from Arthrobacter nicotinovorans and identification of a pAO1-dependent nicotine uptake system.

The 165-kb catabolic plasmid pAO1 enables the gram-positive soil bacterium Arthrobacter nicotinovorans to grow on the tobacco alkaloid L-nicotine. The 165,137-nucleotide sequence, with an overall G+C content of 59.7%, revealed, besides genes and open reading frames (ORFs) for nicotine degradation, a complete set of ORFs for enzymes essential for the biosynthesis of the molybdenum dinucleotide cofactor, as well as ORFs related to uptake and utilization of carbohydrates, sarcosine, and amino acids.

Single-nucleotide polymorphism detection using peptide nucleic acids.

Variations in the human DNA sequence between individuals can be an indication of predisposition to disease, affect the response to drug treatment, or more directly, be the fingerprint of an inheritable trait or defect. Significant efforts at improving the speed, accuracy and sensitivity of detecting such polymorphisms have led to the development of a number of powerful approaches. Sequence-specific base pairing between the strands of DNA, according to the Watson-Crick model, forms the basis of many detection systems.