Crystallization and crystallographic analysis of an Arabidopsis nuclear proteinaceous RNase P

RNase P activity is ubiquitous and involves the 5' maturation of precursor tRNAs. For a long time, it was thought that all RNases P were ribonucleoproteic enzymes. However, the characterization of RNase P in human mitochondria and in plants revealed a novel kind of RNase P composed of protein only, called PRORP for `proteinaceous RNase P'. Whereas in human mitochondria PRORP has two partners that are required for RNase P activity, PRORP proteins are active as single-subunit enzymes in plants. Three paralogues of PRORP are found in Arabidopsis thaliana. PRORP1 is responsible for RNase P in mitochondria and chloroplasts, while PRORP2 and PRORP3 are nuclear enzymes. Here, the purification and crystallization of the Arabidopsis PRORP2 protein are reported. Optimization of the initial crystallization conditions led to crystals that diffracted to 3 Å resolution

tRNAdb 2009: compilation of tRNA sequences and tRNA genes

One of the first specialized collections of nucleic acid sequences in life sciences was the ‘compilation of tRNA sequences and sequences of tRNA genes’ (http://www.trna.uni-bayreuth.de). Here, an updated and completely restructured version of this compilation is presented (http://trnadb.bioinf.uni-leipzig.de). The new database, tRNAdb, is hosted and maintained in cooperation between the universities of Leipzig, Marburg, and Strasbourg. Reimplemented as a relational database, tRNAdb will be updated periodically and is searchable in a highly flexible and user-friendly way. Currently, it contains more than 12 000 tRNA genes, classified into families according to amino acid specificity. Furthermore, the implementation of the NCBI taxonomy tree facilitates phylogeny-related queries. The database provides various services including graphical representations of tRNA secondary structures, a customizable output of aligned or un-aligned sequences with a variety of individual and combinable search criteria, as well as the construction of consensus sequences for any selected set of tRNAs

Mapping of mitochondrial mRNA termini in Arabidopsis thaliana: t-elements contribute to 5′ and 3′ end formation

With CR–RT–PCR as primary approach we mapped the 5′ and 3′ transcript ends of all mitochondrial protein-coding genes in Arabidopsis thaliana. Almost all transcripts analyzed have single major 3′ termini, while multiple 5′ ends were found for several genes. Some of the identified 5′ ends map within promoter motifs suggesting these ends to be derived from transcription initiation while the majority of the 5' termini seems to be generated post-transcriptionally. Assignment of the extremities of 5′ leader RNAs revealed clear evidence for an endonucleolytic generation of the major cox1 and atp9 5′ mRNA ends. tRNA-like structures, so-called t-elements, are associated either with 5′ or with 3′ termini of several mRNAs. These secondary structures most likely act as cis-signals for endonucleolytic cleavages by RNase Z and/or RNase P. Since no conserved sequence motif is evident at post-transcriptionally derived ends, we suggest t-elements, stem–loops and probably complex higher order structures as cis-elements for processing. This analysis provides novel insights into 5′ and 3′ end formation of mRNAs. In addition, the complete transcript map is a substantial and important basis for future studies of gene expression in mitochondria of higher plants

The PREP suite: predictive RNA editors for plant mitochondrial genes, chloroplast genes and user-defined alignments

RNA editing alters plant mitochondrial and chloroplast transcripts by converting specific cytidines to uridines, which usually results in a change in the amino acid sequence of the translated protein. Systematic studies have experimentally identified sites of RNA editing in organellar transcriptomes from several species, but these analyses have not kept pace with rate of genome sequencing. The PREP (predictive RNA editors for plants) suite was developed to computationally predict sites of RNA editing based on the well-known principle that editing in plant organelles increases the conservation of proteins across species. The PREP suite provides predictive RNA editors for plant mitochondrial genes (PREP-Mt), for chloroplast genes (PREP-Cp), and for alignments submitted by the user (PREP-Aln). These servers require minimal input, are very fast, and are highly accurate on all seed plants examined to date. PREP-Mt has proved useful in several research studies and the newly developed PREP-Cp and PREP-Aln servers should be of further assistance for analyses that require knowledge of the location of sites of RNA editing. The PREP suite is freely available at http://prep.unl.edu/

Synthetic polyamines stimulate in vitro transcription by T7 RNA polymerase.

The influence of nine synthetic polyamines on in vitro transcription with T7 RNA polymerase has been studied. The compounds used were linear or macrocyclic tetra- and hexaamine, varying in their size, shape and number of protonated groups. Their effect was tested on different types of templates, all presenting the T7 RNA promoter in a double-stranded form followed by sequences encoding short transcripts (25 to 35-mers) either on single- or double-stranded synthetic oligodeoxyribonucleotides. All polyamines used stimulate transcription of both types of templates at levels dependent on their size, shape, protonation degree, and concentration. For each compound, an optimal concentration could be defined; above this concentration, transcription inhibition occurred. Highest stimulation (up to 12-fold) was obtained by the largest cyclic compound called [38]N6C10.comparative studyjournal articleresearch support, non-u.s. gov't1994 Jul 25importe

Is plant mitochondrial RNA editing a source of phylogenetic incongruence? An answer from in silico and in vivo data sets

<p>Abstract</p> <p>Background</p> <p>In plant mitochondria, the post-transcriptional RNA editing process converts C to U at a number of specific sites of the mRNA sequence and usually restores phylogenetically conserved codons and the encoded amino acid residues. Sites undergoing RNA editing evolve at a higher rate than sites not modified by the process. As a result, editing sites strongly affect the evolution of plant mitochondrial genomes, representing an important source of sequence variability and potentially informative characters.</p> <p>To date no clear and convincing evidence has established whether or not editing sites really affect the topology of reconstructed phylogenetic trees. For this reason, we investigated here the effect of RNA editing on the tree building process of twenty different plant mitochondrial gene sequences and by means of computer simulations.</p> <p>Results</p> <p>Based on our simulation study we suggest that the editing ‘noise’ in tree topology inference is mainly manifested at the cDNA level. In particular, editing sites tend to confuse tree topologies when artificial genomic and cDNA sequences are generated shorter than 500 bp and with an editing percentage higher than 5.0%. Similar results have been also obtained with genuine plant mitochondrial genes. In this latter instance, indeed, the topology incongruence increases when the editing percentage goes up from about 3.0 to 14.0%. However, when the average gene length is higher than 1,000 bp (<it>rps3</it>, <it>matR</it> and <it>atp1</it>) no differences in the comparison between inferred genomic and cDNA topologies could be detected.</p> <p>Conclusions</p> <p>Our findings by the here reported <it>in silico</it> and <it>in vivo</it> computer simulation system seem to strongly suggest that editing sites contribute in the generation of misleading phylogenetic trees if the analyzed mitochondrial gene sequence is highly edited (higher than 3.0%) and reduced in length (shorter than 500 bp).</p> <p>In the current lack of direct experimental evidence the results presented here encourage, thus, the use of genomic mitochondrial rather than cDNA sequences for reconstructing phylogenetic events in land plants.</p

Characteristics and Prediction of RNA Editing Sites in Transcripts of the Moss Takakia lepidozioides Chloroplast

RNA editing in land plant organelles is a process primarily involving the conversion of cytidine to uridine in pre-mRNAs. The process is required for gene expression in plant organelles, because this conversion alters the encoded amino acid residues and improves the sequence identity to homologous proteins. A recent study uncovered that proteins encoded in the nuclear genome are essential for editing site recognition in chloroplasts; the mechanisms by which this recognition occurs remain unclear. To understand these mechanisms, we determined the genomic and cDNA sequences of moss Takakia lepidozioides chloroplast genes, then computationally analyzed the sequences within −30 to +10 nucleotides of RNA editing sites (neighbor sequences) likely to be recognized by trans-factors. As the T. lepidozioides chloroplast has many RNA editing sites, the analysis of these sequences provides a unique opportunity to perform statistical analyses of chloroplast RNA editing sites. We divided the 302 obtained neighbor sequences into eight groups based on sequence similarity to identify group-specific patterns. The patterns were then applied to predict novel RNA editing sites in T. lepidozioides transcripts; ∼60% of these predicted sites are true editing sites. The success of this prediction algorithm suggests that the obtained patterns are indicative of key sites recognized by trans-factors around editing sites of T. lepidozioides chloroplast genes