Natalia E. Broude

Ab initio RNA folding by discrete molecular dynamics: From structure prediction to folding mechanisms

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 A 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 non-hierarchical 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.

Stem-loop oligonucleotides: a robust tool for molecular biology and biotechnology

The specific structural features of stem-loop (hairpin) DNA constructs provide increased specificity of target recognition. Recently, several robust assays have been developed that exploit the potential of structurally constrained oligonucleotides to hybridize with their cognate targets. Here, I review new diagnostic approaches based on the formation of stem-loop DNA oligonucleotides: molecular beacon methodology, suppression PCR approaches and the use of hairpin probes in DNA microarrays. The advantages of these techniques over existing ones for sequence-specific DNA detection, amplification and manipulation are discussed.

Pig kidney Na+,K+-ATPase: Primary structure and spatial organization

(Na+ + K+)‐ATPase α‐Subunit β‐Subunit cDNA nucleotide sequence Primary structure Glycopeptide Transmembrane arrangement

RNA visualization in live bacterial cells using fluorescent protein complementation

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.

Family of human Na+,K+‐ATPase genes Structure of the gene for the catalytic subunit (αIII‐form) and its relationship with structural features of the protein

The primary structure of a gene of the Na+,K+‐ATPase multigenic family in the human genome has been determined. The gene corresponds to a hypothetical αIII‐form of the enzyme catalytic subunit. The gene comprises over 25000 bp, and its protein coding region includes 23 exons and 22 introns. Possible correlation between structural features of the protein and location of introns in the gene are discussed.

The family of human Na+K+‐ATPase genes

The multigene family of human Na,K-ATPase is composed of 5 alpha-subunit genes, 3 of which were shown to encode the functionally active alpha 1, alpha 2 and alpha 3 isoforms of the catalytic subunits. This report describes the isolation, mapping and partial sequencing of the fourth gene (ATP1AL1) that was demonstrated here to be functionally active and expressed in human brain and kidney. Limited DNA sequencing of the ATP1AL1 exons allowed one to suggest that the gene probably encodes a new ion transport ATPase rather than an isoform of the Na,K-ATPase or the closely related H,K-ATPase.

Spatiotemporal patterns and transcription kinetics of induced RNA in single bacterial cells

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.

High polymorphism level of genomic sequences flanking insertion sites of human endogenous retroviral long terminal repeats.

The polymorphism at the multitude of loci adjacent to human endogenous retrovirus long terminal repeats (LTRs) was analyzed by a technique for whole genome differential display based on the PCR suppression effect that provides selective amplification and display of genomic sequences flanking interspersed repeated elements. This strategy is simple, target‐specific, requires a small amount of DNA and provides reproducible and highly informative data. The average frequency of polymorphism observed in the vicinity of the LTR insertion sites was found to be about 12%. The high incidence of polymorphism within the LTR flanks together with the frequent location of LTRs near genes makes the LTR loci a useful source of polymorphic markers for gene mapping.

The family of human Na,K-ATPase genesATP1AL1 gene is transcriptionally competent and probably encodes the related ion transport ATPase

The multigene family of human Na,K‐ATPase is composed of 5 α‐subunit genes, 3 of which were shown to encode the functionally active α1, α2 and α3 isoforms of the catalytic subunit. This report describes the isolation, mapping and partial sequencing of the fourth gene (ATP1AL1) that was demonstrated here to be functionally active and expressed in human brain and kidney. Limited DNA sequencing of theATP1AL1 exons allowed one to suggest that the gene probably encodes a new ion transport ATPase rather than an isoform of the Na,K‐ATPase or the closely related H,K‐ATPase.

Advances in Na+,K+-ATPase studies: from protein to gene and back to protein

Complete primary structures of both subunits of Na+,K+,ATPase from various sources have been established by a combination of the methods for molecular cloning and protein chemistry. The gene family homologous to the α‐subunit cDNA of animal Na+,K+‐ATPases has been found in the human genome. Some genes of this family encode the known isoforms (αI and αII) of the Na+,K+‐ATPase catalytic subunit. The proteins coded by other genes can be either new isoforms of the Na+,K+‐ATPase catalytic subunit or other ion‐transporting ATPases. Expression of the genes of this family proceeds in a tissue‐specific manner and changes during the postnatal development and neoplastic transformation. The complete exon‐intron structure of one of the genes of this family has been established. This gene codes for the form of the catalytic subunit, the existence of which has been unknown. Apparently, all the genes of the discovered family have a similar intron‐exon structure. There is certain correlation between the gene structure and the proposed domain arrangement of the α‐subunit. The results obtained have become the basis for the experiments which prove the existence of the earlier unknown αIII isoform of the Na+,K+‐ATPase catalytic subunit and have made possible the study of its function.