RNA molecules with novel functions have revived interest in the accurate prediction of RNA three-dimensional (3D) structure and folding dynamics. However, existing methods are inefficient in automated 3D structure prediction. Here, we report a robust computational approach for rapid folding of RNA molecules. We develop a simplified RNA model for discrete molecular dynamics (DMD) simulations, incorporating base-pairing and base-stacking interactions. We demonstrate correct folding of 150 structurally diverse RNA sequences. The majority of DMD-predicted 3D structures have <4 Å deviations from experimental structures. The secondary structures corresponding to the predicted 3D structures consist of 94% native base-pair interactions. Folding thermodynamics and kinetics of tRNA Phe , pseudoknots, and mRNA fragments in DMD simulations are in agreement with previous experimental findings. Folding of RNA molecules features transient, non-native conformations, suggesting nonhierarchical RNA folding. Our method allows rapid conformational sampling of RNA folding, with computational time increasing linearly with RNA length. We envision this approach as a promising tool for RNA structural and functional analyses.

We describe a technique for the detection and localization of RNA transcripts in living cells. The method is based on fluorescent-protein complementation regulated by the interaction of a split RNA-binding protein with its corresponding RNA aptamer. In our design, the RNA-binding protein is the eukaryotic initiation factor 4A (eIF4A). eIF4A is dissected into two fragments, and each fragment is fused to split fragments of the enhanced green fluorescent protein (EGFP). Coexpression of the two protein fusions in the presence of a transcript containing eIF4A-interacting RNA aptamer resulted in the restoration of EGFP fluorescence in Escherichia coli cells. We also applied this technique to the visualization of an aptamer-tagged mRNA and 5S ribosomal RNA (rRNA). We observed distinct spatial and temporal changes in fluorescence within single cells, reflecting the nature of the transcript.

We describe here the identification of eight polymorphic microsatellite loci with (CA) n repeats in the Trypanosoma cruzi genome based on the affinity capture of fragments using biotinylated (CA) 12 attached to streptavidincoated magnetic beads. The presence of two peaks in PCR amplification products from individual clones confirmed that T. cruzi is diploid. Hardy-Weinberg and linkage disequilibrium analyses suggested that sexual reproduction is rare or absent and that the population structure is clonal. Several strains, especially those isolated from nonhuman sources, showed more than two alleles in many loci demonstrating that they were multiclonal. The phylogenetic analysis of T. cruzi based on microsatellites revealed a great genetic distance among strains, although the strain dispersion profile in the Wagner network was in general agreement with the species dimorphism found by PCR amplification of the divergent region of the rRNA 24S␣ gene.

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Large-scale analysis of the GC-content distribution at the gene level reveals both common features and basic differences in genomes of different groups of species. Sharp changes in GC content are detected at the transcription boundaries for all species analyzed, including human, mouse, rat, chicken, fruit fly, and worm. However, two substantially distinct groups of GC-content profiles can be recognized: warm-blooded vertebrates including human, mouse, rat, and chicken, and invertebrates including fruit fly and worm. In vertebrates, sharp positive and negative spikes of GC content are observed at the transcription start and stop sites, respectively, and there is also a progressive decrease in GC content from the 5 untranslated region to the 3 untranslated region along the gene. In invertebrates, the positive and negative GC-content spikes at the transcription start and stop sites are preceded by spikes of opposite value, and the highest GC content is found in the coding regions of the genes. Cross-correlation analysis indicates high frequencies of GC-content spikes at transcription start and stop sites. The strong conservation of this genomic feature seen in comparisons of the human͞mouse and human͞rat orthologs, and the clustering of genes with GC-content spikes on chromosomes imply a biological function. The GC-content spikes at transcription boundaries may reflect a general principle of genomic punctuation. Our analysis also provides means for identifying these GC-content spikes in individual genomic sequences.gene clustering ͉ gene ontology ͉ transcription start site ͉ transcription stop site

Fluorescent proteins have proven to be excellent reporters and biochemical sensors with a wide range of applications. In a split form, they are not fluorescent, but their fluorescence can be restored by supplementary protein-protein or protein-nucleic acid interactions that reassemble the split polypeptides. However, in prior studies, it took hours to restore the fluorescence of a split fluorescent protein because the formation of the protein chromophore slowly occurred de novo concurrently with reassembly. Here we provide evidence that a fluorogenic chromophore can self-catalytically form within an isolated N-terminal fragment of the enhanced green fluorescent protein (EGFP). We show that restoration of the split protein fluorescence can be driven by nucleic acid complementary interactions. In our assay, fluorescence development is fast (within a few minutes) when complementary oligonucleotide-linked fragments of the split EGFP are combined. The ability of our EGFP system to respond quickly to DNA hybridization should be useful for detecting the kinetics of many other types of pairwise interactions both in vitro and in living cells.split EGFP ͉ DNA duplex ͉ EGFP reassembly ͉ protein folding ͉ DMD simulations S plit fluorescent proteins are convenient tools to detect specific protein-protein or protein-nucleic acid interactions (1-5). The approach is based on the reassembly of a fluorescent protein from two nonfluorescent fragments driven by additional biomolecular interactions, and it results in restoration of fluorescence. The development of fluorescence, however, usually takes several hours because of the requirement of the de novo formation of the chromophore within the reassembled protein (6). Because this approach provides a slow response, it would clearly be advantageous to accelerate it.A straightforward way to do this would be to use a fragment of a split protein with a preformed chromophore that is not fluorescent per se but is capable of bright fluorescence within a full-size protein. To the best of our knowledge, such a strategy has not been previously accomplished. In this report, we demonstrate the feasibility of an alternative approach based on the nucleic acidsupported fast complementation of EGFP fragments, one of which contains a mature profluorescent chromophore. Results and Discussion Molecular Modeling of Protein Folding: Large EGFP Fragment CanPotentially Form a Chromophore. In this study, we used two fragments of the EGFP, which are linked, in its native structure, by a flexible loop of nine amino acids, residues 153-161 (7, 8). The larger, N-terminal EGFP fragment is known to contain the three amino acids that form a chromophore, which is fluorescent in native, but not in denatured, protein (6, 7). It is also known that this tripeptide chromophore exhibits no fluorescence in a separate large EGFP fragment (2, 4). EGFP chromophore formation is a self-catalytic process requiring correct protein folding (6). We were curious to see whether the N-terminal EGFP fragment (approximately two-third...

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Bacteria have a complex internal organization with specific localization of many proteins and DNA, which dynamically move during the cell cycle and in response to changing environmental stimuli. Much less is known, however, about the localization and movements of RNA molecules. By modifying our previous RNA labeling system, we monitor the expression and localization of a model RNA transcript in live Escherichia coli cells. Our results reveal that the target RNA is not evenly distributed within the cell and localizes laterally along the long cell axis, in a pattern suggesting the existence of ordered helical RNA structures reminiscent of known bacterial cytoskeletal cellular elements.espite their relatively small dimensions, bacterial cells show a remarkable, rich internal subcellular organization that has captured the interest of researchers over the past decade (1-4). Many cytoplasmic and membrane proteins, particularly those involved in cell division, DNA replication, and chromosome segregation, have specific subcellular localizations that can change quickly over time in response to cell cycle progression, motility, and environmental cues. This dynamic and organized behavior is also true for bacterial chromosomal DNA. The use of GFP fusions and in situ fluorescence hybridization (FISH) have shown that every chromosomal locus has a defined subcellular address and is replicated and segregated into the new cell as part of an active and directed process (4, 5). Bacterial plasmids, both low and high copy, also have specific cellular addresses and segregate in a fashion that is unique for a given plasmid (6-8).Little is known, however, about RNA dynamics in bacteria. With the advent of new methods to label RNA in live cells, the transcription kinetics, localization, and movement of RNA in the bacterium Escherichia coli has begun to be discerned only recently (9-13).To understand RNA dynamics in live cells better, it would be useful to develop RNA labeling methods that would allow direct visualization and real-time quantitation of RNAs with low background levels. We recently reported a system based on protein complementation that uses binding of a split and inactive protein complex to a short interacting sequence on a target RNA. The marker protein re-associates and becomes fluorescent only upon binding to RNA (13), which makes this approach more desirable than alternative techniques relying on expression of full-size fluorescent proteins. Briefly, the method consists of fusing the Nterminal fragment of EGFP to the N-terminal domain of an RNA-binding protein, the eukaryotic initiation factor 4A (eIF4A), via a polypeptide linker. Similarly, the C-terminal fragment of EGFP is fused to the C-terminal domain of eIF4A. The target RNA is tagged at the 3Ј end with an aptamer sequence known to bind eIF4A with high affinity (14) (Fig. 1A). Expression of the labeling components in E. coli cells generates a fluorescent signal only in the presence of the target RNA, caused by the reassociation of the two EGFP fragments and forma...