Oleg A. Zverkov

Transcription regulation of plastid genes involved in sulfate transport in Viridiplantae.

This study considers transcription regulation of plastid genes involved in sulfate transport in the parasites of invertebrate (Helicosporidium sp.) and other species of the Viridiplantae. A one-box conserved motif with the consensus TAAWATGATT is found near promoters upstream the cysT and cysA genes in many species. In certain cases, the motif is repeated two or three times.

Plastid-encoded protein families specific for narrow taxonomic groups of algae and protozoa

Protein clustering is useful for refining protein annotations and searching for proteins by their phylogenetic profile. We have performed the clustering of proteins encoded in the plastoms of Rhodophyta, as well as other plastid-containing species related to the Rhodophyta branch. The corresponding database and cluster search according to protein phylogenetic profile are available at http://lab6.iitp.ru/ppc/redline. Plastome-encoded proteins specific for small taxonomic groups of algae and protozoa have been found based on this database, and the search for and analysis of RNA polymerase in the nuclear genomes of Apicomplexa has been performed.

Modeling RNA polymerase competition: the effect of σ-subunit knockout and heat shock on gene transcription level

BackgroundModeling of a complex biological process can explain the results of experimental studies and help predict its characteristics. Among such processes is transcription in the presence of competing RNA polymerases. This process involves RNA polymerases collision followed by transcription termination.ResultsA mathematical and computer simulation model is developed to describe the competition of RNA polymerases during genes transcription on complementary DNA strands. E.g., in the barley Hordeum vulgare the polymerase competition occurs in the locus containing plastome genes psbA, rpl23, rpl2 and four bacterial type promoters. In heat shock experiments on isolated chloroplasts, a twofold decrease of psbA transcripts and even larger increase of rpl23-rpl2 transcripts were observed, which is well reproduced in the model. The model predictions are in good agreement with virtually all relevant experimental data (knockout, heat shock, chromatogram data, etc.). The model allows to hypothesize a mechanism of cell response to knockout and heat shock, as well as a mechanism of gene expression regulation in presence of RNA polymerase competition. The model is implemented for multiprocessor platforms with MPI and supported on Linux and MS Windows. The source code written in C++ is available under the GNU General Public License from the laboratory website. A user-friendly GUI version is also provided at http://lab6.iitp.ru/en/rivals.ConclusionsThe developed model is in good agreement with virtually all relevant experimental data. The model can be applied to estimate intensities of binding of the holoenzyme and phage type RNA polymerase to their promoters using data on gene transcription levels, as well as to predict characteristics of RNA polymerases and the transcription process that are difficult to measure directly, e.g., the intensity (frequency) of holoenzyme binding to the promoter in correlation to its nucleotide composition and the type of σ-subunit, the amount of transcription initiation aborts, etc. The model can be used to make functional predictions, e.g., heat shock response in isolated chloroplasts and changes of gene transcription levels under knockout of different σ-subunits or RNA polymerases or due to gene expression regulation.ReviewersThis article was reviewed by Dr. Anthony Almudevar, Dr. Aniko Szabo, Dr. Yuri Wolf (nominated by Dr. Peter Olofsson) and Prof. Marek Kimmel.

A Database of Plastid Protein Families from Red Algae and Apicomplexa and Expression Regulation of the moeB Gene.

We report the database of plastid protein families from red algae, secondary and tertiary rhodophyte-derived plastids, and Apicomplexa constructed with the novel method to infer orthology. The families contain proteins with maximal sequence similarity and minimal paralogous content. The database contains 6509 protein entries, 513 families and 278 nonsingletons (from which 230 are paralog-free, and among the remaining 48, 46 contain at maximum two proteins per species, and 2 contain at maximum three proteins per species). The method is compared with other approaches. Expression regulation of the moeB gene is studied using this database and the model of RNA polymerase competition. An analogous database obtained for green algae and their symbiotic descendants, and applications based on it are published earlier.

Regulation of Expression and Evolution of Genes in Plastids of Rhodophytic Branch

A novel algorithm and original software were used to cluster all proteins encoded in plastids of 72 species of the rhodophytic branch. The results are publicly available at http://lab6.iitp.ru/ppc/redline72/ in a database that allows fast identification of clusters (protein families) both by a fragment of an amino acid sequence and by a phylogenetic profile of a protein. No such integral clustering with the corresponding functions can be found in the public domain. The putative regulons of the transcription factors Ycf28 and Ycf29 encoded in the plastids were identified using the clustering and the database. A regulation of translation initiation was proposed for the ycf24 gene in plastids of certain red algae and apicomplexans as well as a regulation of a putative gene in apicoplasts of Babesia spp. and Theileria parva. The conserved regulation of the ycf24 gene expression and specificity alternation of the transcription factor Ycf28 were shown in the plastids. A phylogenetic tree of plastids was generated for the rhodophytic branch. The hypothesis of the origin of apicoplasts from the common ancestor of all apicomplexans from plastids of red algae was confirmed.

A method for identification of highly conserved elements and evolutionary analysis of superphylum Alveolata

BackgroundPerfectly or highly conserved DNA elements were found in vertebrates, invertebrates, and plants by various methods. However, little is known about such elements in protists. The evolutionary distance between apicomplexans can be very high, in particular, due to the positive selection pressure on them. This complicates the identification of highly conserved elements in alveolates, which is overcome by the proposed algorithm.ResultsA novel algorithm is developed to identify highly conserved DNA elements. It is based on the identification of dense subgraphs in a specially built multipartite graph (whose parts correspond to genomes). Specifically, the algorithm does not rely on genome alignments, nor pre-identified perfectly conserved elements; instead, it performs a fast search for pairs of words (in different genomes) of maximum length with the difference below the specified edit distance. Such pair defines an edge whose weight equals the maximum (or total) length of words assigned to its ends. The graph composed of these edges is then compacted by merging some of its edges and vertices. The dense subgraphs are identified by a cellular automaton-like algorithm; each subgraph defines a cluster composed of similar inextensible words from different genomes. Almost all clusters are considered as predicted highly conserved elements. The algorithm is applied to the nuclear genomes of the superphylum Alveolata, and the corresponding phylogenetic tree is built and discussed.ConclusionWe proposed an algorithm for the identification of highly conserved elements. The multitude of identified elements was used to infer the phylogeny of Alveolata.

Modeling RNA polymerase interaction in mitochondria of chordates.

BackgroundIn previous work, we introduced a concept, a mathematical model and its computer realization that describe the interaction between bacterial and phage type RNA polymerases, protein factors, DNA and RNA secondary structures during transcription, including transcription initiation and termination. The model accurately reproduces changes of gene transcription level observed in polymerase sigma-subunit knockout and heat shock experiments in plant plastids. The corresponding computer program and a user guide are available at http://lab6.iitp.ru/en/rivals. Here we apply the model to the analysis of transcription and (partially) translation processes in the mitochondria of frog, rat and human. Notably, mitochondria possess only phage-type polymerases. We consider the entire mitochondrial genome so that our model allows RNA polymerases to complete more than one circle on the DNA strand.ResultsOur model of RNA polymerase interaction during transcription initiation and elongation accurately reproduces experimental data obtained for plastids. Moreover, it also reproduces evidence on bulk RNA concentrations and RNA half-lives in the mitochondria of frog, human with or without the MELAS mutation, and rat with normal (euthyroid) or hyposecretion of thyroid hormone (hypothyroid). The transcription characteristics predicted by the model include: (i) the fraction of polymerases terminating at a protein-dependent terminator in both directions (the terminator polarization), (ii) the binding intensities of the regulatory protein factor (mTERF) with the termination site and, (iii) the transcription initiation intensities (initiation frequencies) of all promoters in all five conditions (frog, healthy human, human with MELAS syndrome, healthy rat, and hypothyroid rat with aberrant mtDNA methylation). Using the model, absolute levels of all gene transcription can be inferred from an arbitrary array of the three transcription characteristics, whereas, for selected genes only relative RNA concentrations have been experimentally determined. Conversely, these characteristics and absolute transcription levels can be obtained using relative RNA concentrations and RNA half-lives known from various experimental studies. In this case, the “inverse problem” is solved with multi-objective optimization.ConclusionsIn this study, we demonstrate that our model accurately reproduces all relevant experimental data available for plant plastids, as well as the mitochondria of chordates. Using experimental data, the model is applied to estimate binding intensities of phage-type RNA polymerases to their promoters as well as predicting terminator characteristics, including polarization. In addition, one can predict characteristics of phage-type RNA polymerases and the transcription process that are difficult to measure directly, e.g., the association between the promoter’s nucleotide composition and the intensity of polymerase binding. To illustrate the application of our model in functional predictions, we propose a possible mechanism for MELAS syndrome development in human involving a decrease of Phe-tRNA, Val-tRNA and rRNA concentrations in the cell. In addition, we describe how changes in methylation patterns of the mTERF binding site and three promoters in hypothyroid rat correlate with changes in intensities of the mTERF binding and transcription initiations. Finally, we introduce an auxiliary model to describe the interaction between polysomal mRNA and ribonucleases.

Comparative Analysis of Apicoplast-Targeted Protein Extension Lengths in Apicomplexan Parasites.

In general, the mechanism of protein translocation through the apicoplast membrane requires a specific extension of a functionally important region of the apicoplast-targeted proteins. The corresponding signal peptides were detected in many apicomplexans but not in the majority of apicoplast-targeted proteins in Toxoplasma gondii. In T. gondii signal peptides are either much diverged or their extension region is processed, which in either case makes the situation different from other studied apicomplexans. We propose a statistic method to compare extensions of the functionally important regions of apicoplast-targeted proteins. More specifically, we provide a comparison of extension lengths of orthologous apicoplast-targeted proteins in apicomplexan parasites. We focus on results obtained for the model species T. gondii, Neospora caninum, and Plasmodium falciparum. With our method, cross species comparisons demonstrate that, in average, apicoplast-targeted protein extensions in T. gondii are 1.5-fold longer than in N. caninum and 2-fold longer than in P. falciparum. Extensions in P. falciparum less than 87 residues in size are longer than the corresponding extensions in N. caninum and, reversely, are shorter if they exceed 88 residues.

Gene expression regulation of the PF00480 or PF14340 domain proteins suggests their involvement in sulfur metabolism

The paper studies proteins with domains PF00480 or PF14340, as well as some other poorly characterized proteins, encoded by genes associated with leader peptide genes containing a tract of cysteine codons. Such proteins are hypothetically regulated with cysteine-dependent transcription attenuation, namely the Rho-dependent or classic transcription attenuation. Cysteine is an important structural amino acid in various proteins and is required for synthesis of many sulfur-containing compounds, such as methionine, thiamine, glutathione, taurine and the lipoic acid. Earlier a few species of mycobacteria were predicted by the authors to have cysteine-dependent regulation of operons containing the cysK gene. In Escherichia coli this regulation is absent, and the same operon is regulated by the CysB transcription activator. The paper also studies Rho-dependent and classic transcription regulations in all annotated genes of mycobacteria available in GenBank and their orthologs in Actinomycetales. We predict regulations for many genes involved in sulfur metabolism and transport of sulfur-containing compounds; these regulations differ considerably among species. On the basis of predictions, we assign a putative role to proteins encoded by the regulated genes with unknown function, and also describe the structure of corresponding regulons, predict the lack of such regulations for many genes. Thus, all proteins with the uncharacterized Pfam domains PF14340 and PF00480, as well as some others, are predicted to be involved in sulfur metabolism. We also surmise the affinity of some transporters to sulfur-containing compounds. The obtained results considerably extend earlier large-scale studies of Rho-dependent and classic transcription attenuations.

Study of Direct Repeats in Micro Evolution of Plant Mitochondria and Plastids Based on Protein Clustering

The study focuses on insertions of perfect direct repeats of words of arbitrary length in plastomes and mitochondriomes. The approach is exemplified using seed plants. Plastomes of close species were analyzed to further develop and refine published evidence for the evolution of non-coding DNA. The results suggest that perfect repeats are common elementary events resulting from replication errors—duplication of DNA. The role of such duplications in the evolution of the plastome is discussed.