3,720 research outputs found
ChIP-exo interrogation of Crp, DNA, and RNAP holoenzyme interactions
Numerous in vitro studies have yielded a refined picture of the structural and molecular associations between Cyclic-AMP receptor protein (Crp), the DNA motif, and RNA polymerase (RNAP) holoenzyme. In this study, high-resolution ChIP-exonuclease (ChIP-exo) was applied to study Crp binding in vivo and at genome-scale. Surprisingly, Crp was found to provide little to no protection of the DNA motif under activating conditions. Instead, Crp demonstrated binding patterns that closely resembled those generated by σ70. The binding patterns of both Crp and σ70 are indicative of RNAP holoenzyme DNA footprinting profiles associated with stages during transcription initiation that occur post-recruitment. This is marked by a pronounced advancement of the template strand footprint profile to the +20 position relative to the transcription start site and a multimodal distribution on the nontemplate strand. This trend was also observed in the familial transcription factor, Fnr, but full protection of the motif was seen in the repressor ArcA. Given the time-scale of ChIP studies and that the rate-limiting step in transcription initiation is typically post recruitment, we propose a hypothesis where Crp is absent from the DNA motif but remains associated with RNAP holoenzyme post-recruitment during transcription initiation. The release of Crp from the DNA motif may be a result of energetic changes that occur as RNAP holoenzyme traverses the various stable intermediates towards elongation complex formation
Genome wide prediction of HNF4α functional binding sites by the use of local and global sequence context
An application of machine learning algorithms enables prediction of the functional context of transcription factor binding sites in the human genome
A survey of DNA motif finding algorithms
Background: Unraveling the mechanisms that regulate gene expression is a major challenge in biology. An important task in this challenge is to identify regulatory elements, especially the binding sites in deoxyribonucleic acid (DNA) for transcription factors. These binding sites are short DNA segments that are called motifs. Recent advances in genome sequence availability and in high-throughput gene expression analysis technologies have allowed for the development of computational methods for motif finding. As a result, a large number of motif finding algorithms have been implemented and applied to various motif models over the past decade. This survey reviews the latest developments in DNA motif finding algorithms.Results: Earlier algorithms use promoter sequences of coregulated genes from single genome and search for statistically overrepresented motifs. Recent algorithms are designed to use phylogenetic footprinting or orthologous sequences and also an integrated approach where promoter sequences of coregulated genes and phylogenetic footprinting are used. All the algorithms studied have been reported to correctly detect the motifs that have been previously detected by laboratory experimental approaches, and some algorithms were able to find novel motifs. However, most of these motif finding algorithms have been shown to work successfully in yeast and other lower organisms, but perform significantly worse in higher organisms.Conclusion: Despite considerable efforts to date, DNA motif finding remains a complex challenge for biologists and computer scientists. Researchers have taken many different approaches in developing motif discovery tools and the progress made in this area of research is very encouraging. Performance comparison of different motif finding tools and identification of the best tools have proven to be a difficult task because tools are designed based on algorithms and motif models that are diverse and complex and our incomplete understanding of the biology of regulatory mechanism does not always provide adequate evaluation of underlying algorithms over motif models.Peer reviewedComputer Scienc
A Review on Gene Expression Studies using Microarray
The aim of this review such technologies allow the analysis of different constituents namely the transcripts, proteins and metabolites of the cell that help to deduce gene function. Microarray is one of the technologies permitting simultaneous interrogation of complex nucleic acid mixtures. The genome-wide examination of gene expression with this technology has provided transcript profiles of numerous biological samples for studying population variations, genotype-phenotype relationships, knock-out pools, whole genome genetic foot printing, and genome mismatch scanning. The information obtained by coupling of a microarray expression database and data analysis and viewing tools can serve as a foundation for proposing hypotheses and performing dedicated research. DNA microarray will substantially increase the speed at which differential gene expression can be analyzed and gene functions are elucidated. DNA microarray technology, however, limits expression studies to the mRNA level. Biological processes regulated at RNA degradation, protein synthesis, protein degradation or protein modification will be inert to the microarray approach. Ideally, it should be accompanied by analyses at the protein level. Keyword: Microarray, cDNA, mRNA, genotype-phenotype relationships
Statistical extraction of Drosophila cis-regulatory modules using exhaustive assessment of local word frequency
BACKGROUND: Transcription regulatory regions in higher eukaryotes are often represented by cis-regulatory modules (CRM) and are responsible for the formation of specific spatial and temporal gene expression patterns. These extended, ~1 KB, regions are found far from coding sequences and cannot be extracted from genome on the basis of their relative position to the coding regions. RESULTS: To explore the feasibility of CRM extraction from a genome, we generated an original training set, containing annotated sequence data for most of the known developmental CRMs from Drosophila. Based on this set of experimental data, we developed a strategy for statistical extraction of cis-regulatory modules from the genome, using exhaustive analysis of local word frequency (LWF). To assess the performance of our analysis, we measured the correlation between predictions generated by the LWF algorithm and the distribution of conserved non-coding regions in a number of Drosophila developmental genes. CONCLUSIONS: In most of the cases tested, we observed high correlation (up to 0.6–0.8, measured on the entire gene locus) between the two independent techniques. We discuss computational strategies available for extraction of Drosophila CRMs and possible extensions of these methods
Evolution of MIR168 paralogs in Brassicaceae
<p>Abstract</p> <p>Background</p> <p>In plants, expression of ARGONAUTE1 (AGO1), the catalytic subunit of the RNA-Induced Silencing Complex responsible for post-transcriptional gene silencing, is controlled through a feedback loop involving the miR168 microRNA. This complex auto-regulatory loop, composed of miR168-guided AGO1-catalyzed cleavage of <it>AGO1 </it>mRNA and AGO1-mediated stabilization of miR168, was shown to ensure the maintenance of AGO1 homeostasis that is pivotal for the correct functioning of the miRNA pathway.</p> <p>Results</p> <p>We applied different approaches to studying the genomic organization and the structural and functional evolution of <it>MIR168 </it>homologs in Brassicaeae. A whole genome comparison of Arabidopsis and poplar, phylogenetic footprinting and phylogenetic reconstruction were used to date the duplication events originating <it>MIR168 </it>homologs in these genomes. While orthology was lacking between Arabidopsis and poplar <it>MIR168 </it>genes, we successfully isolated orthologs of both loci present in Arabidopsis (<it>MIR168a </it>and <it>MIR168b</it>) from all the Brassicaceae species analyzed, including the basal species <it>Aethionema grandiflora</it>, thus indicating that (1) independent duplication events took place in Arabidopsis and poplar lineages and (2) the origin of <it>MIR168 </it>paralogs predates both the Brassicaceae radiation and the Arabidopsis alpha polyploidization. Different phylogenetic footprints, corresponding to known functionally relevant regions (transcription starting site and double-stranded structures responsible for microRNA biogenesis and function) or for which functions could be proposed, were found to be highly conserved among <it>MIR168 </it>homologs. Comparative predictions of the identified microRNAs also indicate extreme conservation of secondary structure and thermodynamic stability.</p> <p>Conclusion</p> <p>We used a comparative phylogenetic footprinting approach to identify the structural and functional constraints that shaped <it>MIR168 </it>evolution in Brassicaceae. Although their duplication happened at least 40 million years ago, we found evidence that both <it>MIR168 </it>paralogs have been maintained throughout the evolution of Brassicaceae, most likely functionally as indicated by the extremely high conservation of functionally relevant regions, predicted secondary structure and thermodynamic profile. Interestingly, the expression patterns observed in Arabidopsis indicate that <it>MIR168b </it>underwent partial subfunctionalization as determined by the experimental characterization of its expression pattern provided in this study. We found further evolutionary evidence that pre-miR168 lower stem (the RNA-duplex structure adjacent to the miR-miR* stem) is significantly longer than animal lower stems and probably plays a relevant role in multi-step miR168 biogenesis.</p
Conformational features of the human U2-U6 snRNA complex
The splicing of precursor messenger (pre-m) RNA, during which noncoding intervening sequences are excised and flanking coding regions ligated, is an integral reaction of gene expression. In eukaryotes, it is carried out by a dynamic RNA-protein complex called the spliceosome, in which five small nuclear (sn) RNA components are actively involved in recognition and chemical aspects of the process. A complex formed between U2 and U6 snRNAs is implicated in the chemistry of pre-mRNA splicing. The catalytic activity of the U2-U6 snRNA complex is dependent on the presence of Mg2+ ions, and the complex has been shown to have several specifically bound Mg2+ binding sites in vitro. The overall goal of this research is to characterize the conformational changes of the human U2-U6 snRNA complex upon addition of Mg2+. In order to pursue this question, we attempted to characterize the lowest energy structure of the complex in the absence of spliceosomal proteins using a combination of biophysical and biochemical techniques in the solution state. We first used enzymatic structure probing to evaluate the secondary structural fold of protein-free human U2-U6 snRNA complex. Cleavage patterns resulting from probing reactions were consistent with formation of four stem regions surrounding the junction, therefore favoring the four-helix model consistent with previous results of in vivo studies of the human U2-U6 snRNA complex. However, 19F NMR studies from our laboratory also identified a lesser fraction (up to 14%) of a three- helix conformation. Upon addition of up to 100 mM Mg2+, a slight increase in cleavage by enzymes specific for both single-stranded and double-stranded regions was observed at the junction region, suggesting that this region is becoming more accessible, perhaps because of an increase in the fraction of the three-helix conformation. Analytical ultracentrifugation studies revealed that the Stokes radius of the RNA complex decreased slightly from 31.3 Ã? to 27.9 Ã? in the presence of 100 mM Mg2+, suggesting a slight compaction of the tertiary structure in the presence of divalent metal ions. Hydroxyl radical footprinting experiments on this complex showed signs of increased protection in some areas near and more distant from the junction upon addition of Mg2+, suggesting a change in three-dimensional conformation. Therefore, it appears that Mg2+ induces a small three-dimensional conformational change on human U2-U6 snRNA complex. In order to build a three-dimensional model for the four-helix conformation, we designed a mutant that favors the formation of four-helix conformation and performed SAXS experiments on it. The preliminary SAXS studies suggest that the human U2-U6 snRNA complex and the mutant complex may also be amenable to further study by SAXS. These results act as a good starting point to characterize further the overall global conformation of human U2-U6 snRNA complex and effects of spliceosomal proteins on it
Evaluation of phylogenetic footprint discovery for predicting bacterial cis-regulatory elements and revealing their evolution
The detection of conserved motifs in promoters of orthologous genes (phylogenetic footprints) has become a common strategy to predict cis-acting regulatory elements. Several software tools are routinely used to raise hypotheses about regulation. However, these tools are generally used as black boxes, with default parameters. A systematic evaluation of optimal parameters for a footprint discovery strategy can bring a sizeable improvement to the predictions.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe
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