Crystal structure of Bacillus subtilis TrmB, the tRNA (m(7)G46) methyltransferase

The structure of Bacillus subtilis TrmB (BsTrmB), the tRNA (m(7)G46) methyltransferase, was determined at a resolution of 2.1 Å. This is the first structure of a member of the TrmB family to be determined by X-ray crystallography. It reveals a unique variant of the Rossmann-fold methyltransferase (RFM) structure, with the N-terminal helix folded on the opposite site of the catalytic domain. The architecture of the active site and a computational docking model of BsTrmB in complex with the methyl group donor S-adenosyl-l-methionine and the tRNA substrate provide an explanation for results from mutagenesis studies of an orthologous enzyme from Escherichia coli (EcTrmB). However, unlike EcTrmB, BsTrmB is shown here to be dimeric both in the crystal and in solution. The dimer interface has a hydrophobic core and buries a potassium ion and five water molecules. The evolutionary analysis of the putative interface residues in the TrmB family suggests that homodimerization may be a specific feature of TrmBs from Bacilli, which may represent an early stage of evolution to an obligatory dimer

EgMYB2, a new transcriptional activator from Eucalyptus xylem, regulates secondary cell wall formation and lignin biosynthesis

International audienceEgMYB2, a member of a new subgroup of the R2R3 MYB family of transcription factors, was cloned from a library consisting of RNA from differentiating Eucalyptus xylem. EgMYB2 maps to a unique locus on the Eucalyptus grandis linkage map and co-localizes with a quantitative trait locus (QTL) for lignin content. Recombinant EgMYB2 protein was able to bind specifically the cis-regulatory regions of the promoters of two lignin biosynthetic genes, cinnamoyl-coenzyme A reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD), which contain MYB consensus binding sites. EgMYB2 was also able to regulate their transcription in both transient and stable expression assays. Transgenic tobacco plants over-expressing EgMYB2 displayed phenotypic changes relative to wild-type plants, among which were a dramatic increase in secondary cell wall thickness, and an alteration of the lignin profiles. Transcript abundance of genes encoding enzymes specific to lignin biosynthesis was increased to varying extents according to the position of individual genes in the pathway,whereas core phenylpropanoid geneswere not significantly affected. Together these results suggest a role for EgMYB2 in the co-ordinated control of genes belonging to the monolignol-specific pathway, and therefore in the biosynthesis of lignin and the regulation of secondary cell wall formation

The YqfN protein of Bacillus subtilis is the tRNA: m1A22 methyltransferase (TrmK)

N1-methylation of adenosine to m1A occurs in several different positions in tRNAs from various organisms. A methyl group at position N1 prevents Watson–Crick-type base pairing by adenosine and is therefore important for regulation of structure and stability of tRNA molecules. Thus far, only one family of genes encoding enzymes responsible for m1A methylation at position 58 has been identified, while other m1A methyltransferases (MTases) remain elusive. Here, we show that Bacillus subtilis open reading frame yqfN is necessary and sufficient for N1-adenosine methylation at position 22 of bacterial tRNA. Thus, we propose to rename YqfN as TrmK, according to the traditional nomenclature for bacterial tRNA MTases, or TrMet(m1A22) according to the nomenclature from the MODOMICS database of RNA modification enzymes. tRNAs purified from a ΔtrmK strain are a good substrate in vitro for the recombinant TrmK protein, which is sufficient for m1A methylation at position 22 as are tRNAs from Escherichia coli, which natively lacks m1A22. TrmK is conserved in Gram-positive bacteria and present in some Gram-negative bacteria, but its orthologs are apparently absent from archaea and eukaryota. Protein structure prediction indicates that the active site of TrmK does not resemble the active site of the m1A58 MTase TrmI, suggesting that these two enzymatic activities evolved independently

Fifteen years of research on oral–facial–digital syndromes: from 1 to 16 causal genes

Oral–facial–digital syndromes (OFDS) gather rare genetic disorders characterised by facial, oral and digital abnormalities associated with a wide range of additional features (polycystic kidney disease, cerebral malformations and several others) to delineate a growing list of OFDS subtypes. The most frequent, OFD type I, is caused by a heterozygous mutation in the OFD1 gene encoding a centrosomal protein. The wide clinical heterogeneity of OFDS suggests the involvement of other ciliary genes. For 15 years, we have aimed to identify the molecular bases of OFDS. This effort has been greatly helped by the recent development of whole-exome sequencing (WES). Here, we present all our published and unpublished results for WES in 24 cases with OFDS. We identified causal variants in five new genes (C2CD3, TMEM107, INTU, KIAA0753 and IFT57) and related the clinical spectrum of four genes in other ciliopathies (C5orf42, TMEM138, TMEM231 and WDPCP) to OFDS. Mutations were also detected in two genes previously implicated in OFDS. Functional studies revealed the involvement of centriole elongation, transition zone and intraflagellar transport defects in OFDS, thus characterising three ciliary protein modules: the complex KIAA0753-FOPNL-OFD1, a regulator of centriole elongation; the Meckel-Gruber syndrome module, a major component of the transition zone; and the CPLANE complex necessary for IFT-A assembly. OFDS now appear to be a distinct subgroup of ciliopathies with wide heterogeneity, which makes the initial classification obsolete. A clinical classification restricted to the three frequent/well-delineated subtypes could be proposed, and for patients who do not fit one of these three main subtypes, a further classification could be based on the genotype

Deciduous Trees and the Application of Universal DNA Barcodes: A Case Study on the Circumpolar Fraxinus

The utility of DNA barcoding for identifying representative specimens of the circumpolar tree genus Fraxinus (56 species) was investigated. We examined the genetic variability of several loci suggested in chloroplast DNA barcode protocols such as matK, rpoB, rpoC1 and trnH-psbA in a large worldwide sample of Fraxinus species. The chloroplast intergenic spacer rpl32-trnL was further assessed in search for a potentially variable and useful locus. The results of the study suggest that the proposed cpDNA loci, alone or in combination, cannot fully discriminate among species because of the generally low rates of substitution in the chloroplast genome of Fraxinus. The intergenic spacer trnH-psbA was the best performing locus, but genetic distance-based discrimination was moderately successful and only resulted in the separation of the samples at the subgenus level. Use of the BLAST approach was better than the neighbor-joining tree reconstruction method with pairwise Kimura's two-parameter rates of substitution, but allowed for the correct identification of only less than half of the species sampled. Such rates are substantially lower than the success rate required for a standardised barcoding approach. Consequently, the current cpDNA barcodes are inadequate to fully discriminate Fraxinus species. Given that a low rate of substitution is common among the plastid genomes of trees, the use of the plant cpDNA “universal” barcode may not be suitable for the safe identification of tree species below a generic or sectional level. Supplementary barcoding loci of the nuclear genome and alternative solutions are proposed and discussed

New compounds: peptide derivatives of the antitumor agent N-phosphonoacetyl-L-aspartic acid.

Two peptide forms of the antitumor transition state analogue N-phosphonoacetyl-L-aspartic acid (N2-phosphonoacetyl-N4-glycylglycinamidoethyl-L-asparagine and N1-glycylglycinamidoethyl-N2-phosphonoacetyl-L-isoasparagine ) have been synthesized to obtain potential medicinal agents useful as prodrugs or in a lysosomotropic carrier approach. The bridging unit, ethylenediamine, used for synthetic purposes might be of general interest.Journal Articleinfo:eu-repo/semantics/publishe

Synthesis of a peptide form of N-delta-(phosphonoacetyl)-L-ornithine. Its antibacterial effect through the specific inhibition of Escherichia coli L-ornithine carbamoyltransferase.

N-delta-(Phosphonoacetyl)-L-ornithine is a potent inhibitor of the Escherichia coli L-ornithine carbamoyltransferase (Ki = 0.77 microM, pH 8.0, 37 degrees C). Nevertheless, the analog does not cross the bacterial membrane. Therefore we have designed a tripeptide, glycylglycyl-N-delta-(phosphonoacetyl)-L-ornithine, to take advantage of the broad specificity of the oligopeptide permease system of the bacterium. A lag effect, related to the tripeptide concentration, was observed in the growth of the wild type P4X strain. At high concentration (greater than or equal to 0.75 mM) the peptide appears to be bacteriostatic and the cells which escape this action were characterized gentically as mutants devoid of the oligopeptide transport system. It was shown that the in vivo cellular target of the toxic tripeptide is solely restricted to L-ornithine carbamoyl-transferase and that the tripeptide is probably split in the cell to permit an effective inhibition by N-delta-(phosphonoacetyl)-L-ornithine. Resistance of the wild type cells to moderate levels (less than 0.75 mM) of the phosphonic analog is accompanied by a derepression of the L-ornithine carbamoyltransferase activity. The P4XB2 strain, which is an arg R regulatory mutant, has a reduced lag effect in the presence of the tripeptide and appears to react to the intoxication by a further adjustment of the L-ornithine carbamoyltransferase cellular level.Journal Articleinfo:eu-repo/semantics/publishe

Pathways of glutathione degradation in the yeast Saccharomyces cerevisiae

The degradation of glutathione (GSH) in the yeast Saccharomyces cerevisiae appears to be mediated only by γ-glutamyltranspeptidase and cysteinylglycine dipeptidase. Other enzymes of the γ-glutamyl cycle, γ-glutamyl cyclotransferase and 5-oxo-l-prolinase, are not present in the yeast. In vivo transpeptidation was shown in the presence of a high intracellular level of γ-glutamyltranspeptidase, but only when the de-repressing nitrogen source was a suitable acceptor of the transferase reaction. In contrast, when the de-repressing source was not an acceptor of the transferase reaction (e.g. urea), only glutamate was detected. Intracellular GSH is virtually inert when the level of γ-glutamyltranspeptidase is low. Possible roles for in vivo transpeptidation are discussed. © 1985.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Repression of Escherichia coli carbamoylphosphate synthase: relationships with enzyme synthesis in the arginine and pyrimidine pathways

Cumulative repression of E. coli carbamoylphosphate synthase (CPSase; EC 2.7.2.9) by arginine and pyrimidine was analyzed in relation to control of enzyme synthesis in the arginine and pyrimidine pathways. The expression of carA and carB, the adjacent genes that specify the two subunits of the enzyme, was estimated by means of an in vitro complementation assay. The synthesis of each gene product was found to be under repression control. Coordinate expression of the two genes was observed under most conditions investigated. They might thus form an operon. The preparation of strains blocked in the degradation of cytidine and harboring leaky mutations affecting several steps of pyrimidine nucleotide synthesis made it possible to distinguish between the effects of cytidine and uridine compounds in the repression of the pyrimidine pathway enzymes. The data obtained suggest that derivatives of both cytidine and uridine participate in the repression of CPSase. In addition, repression of CPSase by arginine did not appear to occur unless pyrimidines were present at a significant intracellular concentration. This observation, together with our previous report that argR mutations impair the cumulative repression of CPSase, suggests that this control is mediated through the concerted effects of regulatory elements specific for the arginine and pyrimidine pathways.SCOPUS: ar.jinfo:eu-repo/semantics/publishe