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At least 307 records · Page 17Linked to original sources
PubMed2003

Spectroscopic and thermodynamic studies on the binding of sanguinarine and berberine to triple and double helical DNA and RNA structures.

A comparative study on the interaction of sanguinarine and berberine with DNA and RNA triplexes and their parent duplexes was performed, by using a combination of spectrophotometric, UV thermal melting, circular dichroic and thermodynamic techniques. Formation of the DNA and RNA triplexes was confirmed from UV-melting and circular dichroic measurements. The interaction process was characterized by increase of thermal melting temperature, perturbation in circular dichroic spectrum and the typical hypochromic and bathochromic effects in the absorption spectrum. Scatchard analysis indicated that both the alkaloids bound to the triplex and duplex structures in a non-cooperative manner and the binding was stronger to triplexes than to parent duplexes. Thermal melting studies further indicated that sanguinarine stabilized the Hoogsteen base paired third strand of both DNA and RNA triplexes more tightly compared to their Watson-Crick strands, while berberine stabilized the third strand only without affecting the Watson-Crick strand. However, sanguinarine stabilized the parent duplexes while no stabilization was observed with berberine under identical conditions. Circular dichroic studies were also consistent with the observation that perturbations of DNA and RNA triplexes were more compared to their parent duplexes in presence of the alkaloids. Thermodynamic data revealed that binding of sanguinarine and berberine to triplexes (T.AxT and U.AxU) and duplexes (A.T and A.U) showed negative enthalpy changes and positive entropy changes but that of sanguinarine to C.GxC(+) triplex and G.C duplex exhibited negative enthalpy and negative entropy changes. Taken together, these results suggest that both sanguinarine and berberine can bind and stabilize the DNA and RNA triplexes more strongly than their respective parent duplexes.

Alkaloids↗
PubMed1997

Altered 3'-terminal RNA structure in phage Qbeta adapted to host factor-less Escherichia coli.

The RNA phage Qbeta requires for the replication of its genome an RNA binding protein called Qbeta host factor or Hfq protein. Our previous results suggested that this protein mediates the access of replicase to the 3'-end of the Qbeta plus strand RNA. Here we report the results of an evolutionary experiment in which phage Qbeta was adapted to an Escherichia coli Q13 host strain with an inactivated host factor (hfq) gene. This strain initially produced phage at a titer approximately 10,000-fold lower than the wild-type strain and with minute plaque morphology, but after 12 growth cycles, phage titer and plaque size had evolved to levels near those of the wild-type host. RNAs isolated from adapted Qbeta mutants were efficient templates for replicase without host factor in vitro. Electron microscopy showed that mutant RNAs, in contrast to wild-type RNA, efficiently interacted with replicase at the 3'-end in the absence of host factor. The same set of four mutations in the 3'-terminal third of the genome was found in several independently evolved phage clones. One mutation disrupts the base pairing of the 3'-terminal CCCOH sequence, suggesting that the host factor stimulates activity of the wild-type RNA template by melting out its 3'-end.

Base Sequence↗
PubMed1989

A measles virus subgenomic RNA: structure and generation mechanism.

A plaque-derived line of measles virus induced the synthesis of 21S subgenomic RNA in addition to 50S genomic RNA. The nucleotide sequence of the former RNA was determined in comparison with that of the latter. The 21S subgenomic RNA was 2646 nucleotides long and had a copyback structure containing a 50S genomic 5'-end. At the site where copying turns back, there was a 15-nucleotide-long sequence resembling the "measles virus consensus sequence" which is present at the beginning of each gene on the measles virus genome and is presumed to serve as a signal for the initiation of transcription. Recognition of the specific sequence may have been involved in the aberrant replication and may represent an additional and novel copy choice mechanism leading to the generation of subgenomic RNAs.

Base Sequence↗
PubMed1996

A specific RNA structural motif mediates high affinity binding by the HIV-1 nucleocapsid protein (NCp7).

Current research indicates that the nucleocapsid protein (NCp7) of human immunodeficiency virus type 1 (HIV-1) interacts with a variety of RNA substrates during the progression of the viral life cycle. The RNA features specifically recognized by the protein, however, have yet to be identified. SELEX was used to generate a set of RNAs whose affinities for nucleocapsid were on the order of 2 x 10(-9) M. Comparative analysis revealed that each RNA contains a highly conserved fourteen nucleotide sequence-block. Computer modeling and structure probing experiments indicate that the RNA ligands use the consensus sequence to fold into hairpins with an identical asymmetric bulge. The presence of the nucleocapsid protein protects the asymmetric bulge from ribonuclease attack, suggesting that it is the key element in protein recognition. A search for similar structural motifs within the HIV genome reveals several potential interaction sites for the nucleocapsid protein.

Base Sequence↗
PubMed2004

Contributions of the RNA-binding and linker domains and RNA structure to the specificity and affinity of the nucleolin RBD12/NRE interaction.

Nucleolin is a multidomain phosphoprotein involved in ribosome biogenesis. In vitro selection and binding studies with pre-rRNA fragments have shown that the first two RNA-binding domains (RBDs) in nucleolin (RBD12) recognize the consensus sequence (U/G)CCCG(A/G) in the context of a stem-loop structure (nucleolin-recognition element = NRE). Structural studies of nucleolin RBD12 in complex with an in vitro selected NRE (sNRE) and a natural pre-rRNA NRE (b2NRE) have revealed that sequence-specific binding of the consensus NRE is achieved in a similar manner in both complexes using residues in both RBDs as well as the linker connecting them. Using fluorescence anisotropy (FA) and nuclear magnetic resonance (NMR), we demonstrate the importance of the linker for NRE affinity by showing that only the individual RBDs with the linker attached retain the ability to specifically bind, albeit weakly, to sNRE and b2NRE. Binding of RBD1 and RBD2 to the NREs in trans is not detected even when one of the RBDs has the linker attached, which suggests that the linker also contributes to the affinity by tethering the two RBDs. To determine if binding of nucleolin RBD12 to natural NREs is dependent on a specific RNA stem-loop structure, as was the case for the sNRE, we conducted FA and NMR binding assays with nucleolin RBD12 and a single-stranded NRE. The results show that nucleolin RBD12 sequence-specifically binds a single-stranded NRE with an affinity similar to that for b2NRE, indicating that a stem-loop structure is not required for the nucleolin RBD12/pre-rRNA NRE interaction.

Amino Acid Sequence↗
PubMed1997

Recent solution structures of RNA and its complexes with drugs, peptides and proteins.

The past two years have seen remarkable progress in the study of RNA structure: the predicted era of RNA structural biology has arrived. Crystallographic structures of the hammerhead ribozyme and of a large subunit of a group I self-splicing intron have begun to reveal the structural basis of RNA enzymatic activity. A remarkable number of structures of small RNAs and of complexes with drugs, peptides and one protein domain have been determined by NMR.

Forecasting↗
PubMed1986

Analysis of the RNA structural elements involved in the binding of the transcription factor III A from Xenopus laevis.

Xenopus laevis 5S rRNA isolated from 7S particles or transcribed in vitro is found to adopt two alternative conformations. These two conformers contain different structural elements within the major TF III A binding domain, which, when isolated as RNA fragments, still interact with the transcription factor. Chemical modification of easily accessible adenines in their N-1 position does not have any measurable effect on the binding of 5S rRNA to TF III A. These observations are in support of the idea that only a small amount of conserved sequence information is required for the binding of the transcription factor, whereas specific secondary structure features seem to be essential.

Animals↗
PubMedDate not supplied

Use of avian myeloblastosis virus reverse transcriptase at high temperature for sequence analysis of highly structured RNA.

Avian myeloblastosis virus reverse transcriptase (AMV RT) is routinely used in the sequence analysis of RNA and DNA templates. We review the various methods for dealing with secondary structures that would otherwise result in premature termination or sequence compression. Based on our experience in sequencing the 11-kb single-stranded RNA genome of lymphocytic choriomeningitis virus, we have found that raising the reaction temperature above 47 degrees C is the simplest way to overcome template secondary structure, and the use of 98% formamide gels is the simplest way to overcome product secondary structure.

Avian Leukosis Virus↗
PubMed2001

Temporal translational control by a metastable RNA structure.

Programmed cell death by the hok/sok locus of plasmid R1 relies on a complex translational control mechanism. The highly stable hok mRNA is activated by 3'-end exonucleolytical processing. Removal of the mRNA 3' end releases a 5'-end sequence that triggers refolding of the mRNA. The refolded hok mRNA is translatable but can also bind the inhibitory Sok antisense RNA. Binding of Sok RNA leads to irreversible mRNA inactivation by an RNase III-dependent mechanism. A coherent model predicts that during transcription hok mRNA must be refractory to translation and antisense RNA binding. Here we provide genetic evidence for the existence of a 5' metastable structure in hok mRNA that locks the nascent transcript in an inactive configuration in vivo. Consistently, the metastable structure reduces the rate of Sok RNA binding and completely blocks hok translation in vitro. Structural analyses of native RNAs strongly support that the 5' metastable structure exists in the nascent transcript. Further structural analyses reveal that the mRNA 3' end triggers refolding of the mRNA 5' end into the more stable tac-stem conformation. These results provide a profound understanding of an unusual and intricate post-transcriptional control mechanism.

Bacterial Proteins↗
PubMed2004

Conservation of RNA structures enables TNV and BYDV 5' and 3' elements to cooperate synergistically in cap-independent translation.

The subgenomic RNA 2 of tobacco necrosis virus A (TNV sgRNA2) encodes the viral coat protein, is unpolyadenylated and presumably uncapped. Here, we show that TNV sgRNA2 is translated cap independently. This cap-independent translation requires the leader and a 140 nt element of the trailer both in wheat germ extract and in tobacco protoplasts. Similar to barley yellow dwarf virus (BYDV), the TNV 5' and 3' elements stimulate translation synergistically. Computer-aided phylogenetic analysis of the secondary structure of the TNV trailer revealed that the 3' translation element is part of a major conserved stem-loop that contains similarities to structures in the BYDV 3' translation element. These data suggest that the translation mechanisms of TNV sgRNA2 and BYDV RNA are related. To further characterize this relationship, we tested whether cooperativity exists between TNV sgRNA2 and BYDV 5' and 3' elements. We found that the TNV sgRNA2 5' element stimulates translation synergistically with the BYDV 3' element in vitro. This finding is the first evidence for conservation of structures that enable a 5'-3' interaction stimulating cap-independent translation.

3' Untranslated Regions↗
PubMed2001

Satellite tobacco mosaic virus RNA: structure and implications for assembly.

The initial appearance of 45% of the single-stranded RNA of satellite tobacco mosaic virus in electron density maps suggested the entire RNA conformation could be delineated. Subsequent work has localized nearly 80% of the RNA as stem-loop elements. Connection of the stem-loops in the most efficient manner produces a persuasive model for the encapsidated RNA. This arrangement has significant implications for virus assembly and for the essential role of RNA.

Computer Simulation↗
PubMed1983

Loci for human U1 RNA: structural and evolutionary implications.

Three clones U1-1, U1-6, and U1-8 containing sequences related to human U1 RNA have been studied by sequence analysis. The results show that each of the three clones represents a distinct locus. The U1-6 locus is closely related to the HU1-1 locus, which is believed to represent a functional U1 gene. The U1-1 and U1-8 loci are pseudogenes by definition, since they contain sequences that are closely related to but not identical with the human U1 RNA sequence. The U1-6 locus contains the sequence T-A-T-A-T close to the 5'-end of the U1 sequence but it is unclear if this represents the promoter. When the U1-8 locus was compared to the U1-6 locus, it was observed that the 5'-flanking sequences, except in the immediate vicinity of the pseudogene, are as well-conserved as the U1-related sequence itself, at least up to position -220. The high degree of homology in the 5'-flanking region suggests that U1 genes have a much more strict sequence requirement with regard to 5'-flanking sequences than most other eukaryotic genes. The U1-6 and U1-8 loci contain the sequence T-A-T-G-T-A-G-A-T-G-A between positions -211 and -221. An identical sequence is present in the equivalent position in the HU1-1 locus, and may represent the promoter. The high degree of conservation in the postulated promoter region indicates that pseudogenes like U1-8 possibly could be expressed. A truncated U1-related sequence is present between 106 to 150 nucleotides upstream from the U1 gene/pseudogene in the U1-6, the U1-8 and the HU1-1 loci, suggesting that the U1 genes may have been clustered early in evolution. The U1-1 locus has a strikingly different structure from the U1-8 locus; the pseudogene itself is as closely related to the U1 RNA sequence as is the U1-8 pseudogene but the flanking sequences, both on the 5' and the 3' side, share no detectable homology with the corresponding regions in the U1-6 or U1-8 loci. It may therefore be postulated that small nuclear RNA pseudogenes are created by several different mechanisms.

Base Sequence↗
PubMed2005

Solution RNA structures of the HIV-1 dimerization initiation site in the kissing-loop and extended-duplex dimers.

Dimer formation of HIV-1 genomic RNA through its dimerization initiation site (DIS) is crucial to maintaining infectivity. Two types of dimers, the initially generated kissing-loop dimer and the subsequent product of the extended-duplex dimer, are formed in the stem-bulge-stem region with a loop including a self-complementary sequence. NMR chemical shift analysis of a 39mer RNA corresponding to DIS, DIS39, in the kissing-loop and extended-duplex dimers revealed that the three dimensional structures of the stem-bulge-stem region are extremely similar between the two types of dimers. Therefore, we designed two shorter RNA molecules, loop25 and bulge34, corresponding to the loop-stem region and the stem-bulge-stem region of DIS39, respectively. Based upon the chemical shift analysis, the conformation of the loop region of loop25 is identical to that of DIS39 for each of the two types of dimers. The conformation of bulge34 was also found to be the same as that of the corresponding region of DIS39. Thus, we determined the solution structures of loop25 in each of the two types of dimers as well as that of bulge34. Finally, the solution structures of DIS39 in the kissing-loop and extended-duplex dimers were determined by combining the parts of the structures. The solution structures of the two types of dimers were similar to each other in general with a difference found only in the A16 residue. The elucidation of the structures of DIS39 is important to understanding the molecular mechanism of the conformational dynamics of viral RNA molecules.

Base Sequence↗
PubMed2001

The RNA structures engaged in replication and transcription of the A59 strain of mouse hepatitis virus.

In addition to the RI (replicative intermediate RNA) and native RF (replicative form RNA), mouse hepatitis virus-infected cells contained six species of RNA intermediates active in transcribing subgenomic mRNA. We have named these transcriptive intermediates (TIs) and native transcriptive forms (TFs) because they are not replicating genome-sized RNA. Based on solubility in high salt solutions, approximately 70% of the replicating and transcribing structures that accumulated in infected cells by 5-6 h post-infection were multi-stranded intermediates, the RI/TIs. The other 30% were in double-stranded structures, the native RF/TFs. These replicating and transcribing structures were separated by velocity sedimentation on sucrose gradients or by gel filtration chromatography on Sepharose 2B and Sephacryl S-1000, and migrated on agarose gels during electrophoresis, according to their size. Digestion with RNase T1 at 1-10 units/microgram RNA resolved RI/TIs into RF/TF cores and left native RF/TFs intact, whereas RNase A at concentrations of 0.02 microgram/microgram RNA or higher degraded both native RF/TFs and RI/TIs. Viral RI/TIs and native RF/TFs bound to magnetic beads containing oligo(dT)(25), suggesting that the poly(A) sequence on the 3' end of the positive strands was longer than any poly(U) on the negative strands. Kinetics of incorporation of [(3)H]uridine showed that both the RI and TIs were transcriptionally active and the labelling of RI/TIs was not the dead-end product of aberrant negative-strand synthesis. Failure originally to find TIs and TF cores was probably due to overdigestion with RNase A.

Animals↗
PubMed2005

An intricate RNA structure with two tRNA-derived motifs directs complex formation between yeast aspartyl-tRNA synthetase and its mRNA.

Accurate translation of genetic information necessitates the tuned expression of a large group of genes. Amongst them, controlled expression of the enzymes catalyzing the aminoacylation of tRNAs, the aminoacyl-tRNA synthetases, is essential to insure translational fidelity. In the yeast Saccharomyces cerevisiae, expression of aspartyl-tRNA synthetase (AspRS) is regulated in a process necessitating recognition of the 5' extremity of AspRS messenger RNA (mRNA(AspRS)) by its translation product and adaptation to the cellular tRNA(Asp) concentration. Here, we have established the folding of the approximately 300 nucleotides long 5' end of mRNA(AspRS) and identified the structural signals involved in the regulation process. We show that the regulatory region in mRNA(AspRS) folds in two independent and symmetrically structured domains spaced by two single-stranded connectors. Domain I displays a tRNA(Asp) anticodon-like stem-loop structure with mimics of the aspartate identity determinants, that is restricted in domain II to a short double-stranded helix. The overall mRNA structure, based on enzymatic and chemical probing, supports a three-dimensional model where each monomer of yeast AspRS binds one individual domain and recognizes the mRNA structure as it recognizes its cognate tRNA(Asp). Sequence comparison of yeast genomes shows that the features within the mRNA recognized by AspRS are conserved in different Saccharomyces species. In the recognition process, the N-terminal extension of each AspRS subunit plays a crucial role in anchoring the tRNA-like motifs of the mRNA on the synthetase.

Aspartate-tRNA Ligase↗
PubMed2001

HyPaLib: a database of RNAs and RNA structural elements defined by hybrid patterns.

The database, called HyPaLib (for Hybrid Pattern Library), contains annotated structural elements characteristic for certain classes of structural and/or functional RNAs. These elements are described in a language specifically designed for this purpose. The language allows convenient specification of hybrid patterns, i.e. motifs consisting of sequence features and structural elements together with sequence similarity and thermodynamic constraints. We are currently developing software tools that allow a user to search sequence databases for any pattern in HyPaLib, thus providing functionality which is similar to PROSITE, but dedicated to the more complex patterns in RNA sequences. HyPaLib is available at http://bibiserv. techfak.uni-bielefeld.de/HyPa/.

Algorithms↗