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PubMed2003

3DNA: a software package for the analysis, rebuilding and visualization of three-dimensional nucleic acid structures.

We present a comprehensive software package, 3DNA, for the analysis, reconstruction and visualization of three-dimensional nucleic acid structures. Starting from a coordinate file in Protein Data Bank (PDB) format, 3DNA can handle antiparallel and parallel double helices, single-stranded structures, triplexes, quadruplexes and other complex tertiary folding motifs found in both DNA and RNA structures. The analysis routines identify and categorize all base interactions and classify the double helical character of appropriate base pair steps. The program makes use of a recently recommended reference frame for the description of nucleic acid base pair geometry and a rigorous matrix-based scheme to calculate local conformational parameters and rebuild the structure from these parameters. The rebuilding routines produce rectangular block representations of nucleic acids as well as full atomic models with the sugar-phosphate backbone and publication quality 'standardized' base stacking diagrams. Utilities are provided to locate the base pairs and helical regions in a structure and to reorient structures for effective visualization. Regular helical models based on X-ray diffraction measurements of various repeating sequences can also be generated within the program.

Base Pairing↗
PubMedDate not supplied

[mRNA-FAST (mRNA-Function, Activity, STructure) computer system].

Computer system mRNA-FAST (mRNA--Function, Activity, STructure; http://wwwmgs.bionet.nsc.ru/mgs/dbases/trsig/) is described. The system has been developed to analyze nucleotide sequences of mRNA and to measure their essential properties. The system compiles the data base on translation signals including nucleotide sequences of the regulatory regions with structural and experimental information on their specific activities. It also contains programs to search for local homology between mRNA and translation signals, to search for potential signals basing on analysis of the oligonucleotide dictionaries, and to model secondary RNA structure. Possible applications of the system mRNA-FAST are discussed.

Base Sequence↗
PubMed2006

A hexapod nuclear SSU rRNA secondary-structure model and catalog of taxon-specific structural variation.

RNA molecules and in particular the nuclear SSU RNA play an important role in molecular systematics. With the advent of increasingly parameterized substitution models in systematic research, the incorporation of secondary-structure information became a realistic option compensating interdependence of character variation. As a prerequisite, consensus structures of eukaryotic SSU RNA molecules have become available through extensive comparative analyses and crystallographic studies. Despite extensive research in hexapod phylogenetics, consensus SSU RNA secondary structures focusing on hexapods have not yet been explored. In this study, we compiled a representative hexapod SSU data set of 261 sequences and inferred a specific consensus SSU secondary-structure model. Our search for conserved structural motives relied on a combined approach of thermodynamic and covariation analyses. The hexapod consensus-structure model deviates from the canonical eukaryotic model in a number of helices. Additionally, in several helices the hexapod sequences did not support a single consensus structure. We provide consensus structures of these sections of single less-inclusive taxa, thus facilitating the adaptation of the consensus hexapod model to less-inclusive phylogenetic questions. The secondary-structure catalog will foster the application of RNA structure models in phylogenetic analyses using the SSU rRNA molecule, and it will improve the realism of substitution models and the reliability of reconstructions based on rRNA sequences.

Animals↗
PubMed2004

Determinants of apical loop-internal loop RNA-RNA interactions involving the HCV IRES.

Domain II of the hepatitis C virus internal ribosome entry site is a major RNA structure involved in the viral mRNA translation. It comprises four different structural domains. We performed in vitro selection against the apical loop of the domain II and we identified RNA aptamers folding as an imperfect hairpin with an internal loop of interacting with the apical loop of the domain II. This RNA-RNA interaction creates apical loop-internal loop complex. The aptamer binds the target with an apparent K(d) of 35nM. In this study, the main structural elements of the target and the aptamer involved in the formation of the complex are characterized by mutation, deletion, and RNase probing analysis. We demonstrate that a complementary loop flanked by G,C rich upper and lower stems are crucial for such RNA-RNA interactions.

Base Sequence↗
PubMed1995

Deamination of mammalian glutamate receptor RNA by Xenopus dsRNA adenosine deaminase: similarities to in vivo RNA editing.

Double-stranded RNA (dsRNA) adenosine deaminase (dsRAD) converts adenosines to inosines within dsRNA. A great deal of evidence suggests that dsRAD or a related enzyme edits mammalian glutamate receptor mRNA in vivo. Here we map the deamination sites that occur in a truncated glutamate receptor-B (gluR-B) mRNA after incubation with pure Xenopus dsRAD. We find remarkable similarities, as well as distinct differences, between the observed deamination sites and the sites reported to be edited within RNAs isolated from mammalian brain. For example, although deamination at the biologically relevant Q/R editing site occurs, it occurs much less frequently than editing at this site in vivo. We hypothesize that the similarities between the deamination and editing patterns exist because the deamination specificity that is intrinsic to dsRAD is involved in selecting editing sites in vivo. We propose that the observed differences are due to the absence of accessory factors that play indirect roles in vivo, such as binding to and occluding certain sites from dsRAD, or promoting the RNA structure required for correct and efficient editing. The work reported here also suggests that dsRAD is capable of much more selectivity than previously thought; a minimal number of deamination sites (average < or = 5) were found in each gluR-B RNA. We speculate that the observed selectivity is due to the various structural elements (mismatches, bulges, loops) that periodically interrupt the base paired region required for editing.

Adenosine Deaminase↗
PubMed2005

Structure of an RNA polymerase II-RNA inhibitor complex elucidates transcription regulation by noncoding RNAs.

The noncoding RNA B2 and the RNA aptamer FC bind RNA polymerase (Pol) II and inhibit messenger RNA transcription initiation, but not elongation. We report the crystal structure of FC(*), the central part of FC RNA, bound to Pol II. FC(*) RNA forms a double stem-loop structure in the Pol II active center cleft. B2 RNA may bind similarly, as it competes with FC(*) RNA for Pol II interaction. Both RNA inhibitors apparently prevent the downstream DNA duplex and the template single strand from entering the cleft after DNA melting and thus interfere with open-complex formation. Elongation is not inhibited, as nucleic acids prebound in the cleft would exclude the RNA inhibitors. The structure also indicates that A-form RNA could interact with Pol II similarly to a B-form DNA promoter, as suggested for the bacterial transcription inhibitor 6S RNA.

Base Sequence↗
PubMed1996

A characteristic bent conformation of RNA pseudoknots promotes -1 frameshifting during translation of retroviral RNA.

The structures of four different RNA pseudoknots that provide one of the signals required for ribosomal frameshifting in mouse mammary tumor virus have been determined by NMR. The RNA pseudoknots have similar sequences and assume similar secondary structures, but show significantly different frameshifting efficiencies. The three-dimensional structures of one frameshifting and one non-frameshifting RNA pseudoknot had been determined previously by our group. Here we determine the structures of two new RNA pseudoknots, and relate the structures of all four pseudoknots to their frameshifting abilities. The two efficient frameshifting pseudoknots adopt characteristic bent conformations with stem 1 bending towards the major groove of stem 2. In contrast, the two poor frameshifting pseudoknots have structures very different from each other and from the efficient frameshifters. One has linear, coaxially stacked stems, the other has stems twisted and bent, but in the opposite direction to the efficient frameshifters. Changes in loop size that favor bending (shorter loops) increase frameshifting efficiency; longer loops that allow linear arrangement of the stems decrease frameshifting. Frameshifting pseudoknots in feline immunodeficiency virus and simian retrovirus have different loop sequences, but the sequences at their stem junctions imply the same bent conformation as in the mouse mammary tumor viral RNA. The requirement for a precise pseudoknot conformation for efficient frameshifting strongly implies that a specific interaction occurs between the viral RNA pseudoknot and the host protein-synthesizing machinery.

Base Sequence↗
PubMed2003

Nanometer distance measurements on RNA using PELDOR.

A rapid increase in RNA structural studies has been seen in the past few years, in part due to the interest in the multitude of cellular functions performed by RNA. Electron paramagnetic resonance spectroscopy is a useful technique for studying biopolymer structure under physiologically relevant conditions. We present herein the use of pulsed electron double resonance for the determination of a 35 +/- 2 A distance between two spin label nitroxides that were linked to specific positions within an RNA.

Electron Spin Resonance Spectroscopy↗
PubMed1991

Self-cleavage of a 71 nucleotide-long ribozyme derived from hepatitis delta virus genomic RNA.

Self-cleavage efficiency of ribozymes derived from hepatitis delta virus (HDV) has been shown to depend on the RNA structure, which in turn may be determined by the length of the considered sequences. Here we describe the construction and functional analysis of a 71 nucleotide-long RNA genomic fragment, Rz71, which carries an 18 nucleotide deletion in a very stable GC-rich stem-loop (stem IV), predicted to be present in several computer-derived secondary structure models. Rz71 is able to undergo self-cleavage under non-denaturing conditions (the t1/2 of the reaction at 37 degrees C is 3 min). The deletion, however, is not neutral, since under the same conditions the non-deleted ribozyme cleaves to 50% in less than 15 sec. Therefore, stem-loop IV seems to play a structural role, not being directly involved in the catalytic reaction, but contributing to the correct positioning of the catalytic core of the HDV ribozyme. Rz71 is the smallest self-cleaving sub-fragment of HDV genomic RNA reported so far.

Base Sequence↗
PubMedDate not supplied

[Physico-chemical properties of the RNA of Sendai virus. III. Interaction with proflavin].

The binding isotherms of the Sendai virus single- and double-stranded RNA-proflavine complexes have been studied. The existence of two regions on the binding curves, corresponding to two subtypes of the strong complex (I1 and I2) has been demonstrated. The association constants and the numbers of binding sites for both subtypes were determined as a function of ionic strength. Both of the single- and for the double-stranded RNA the association constant for I1 complex were higher than those for I2 complex under all ionic strengths conditions. For the double-stranded RNA the variation of the ionic strength is more importance in case of the I1 complex formation. The total number of binding sites increases with a decrease of ionic strength. At low ionic strength (10(-4) M NaCl) the number of binding sites for single- and double-stranded RNA is practically the same and is equal to the number of binding sites for DNA at high ionic strength (1 molecule of proflavine per 3 nucleotides pairs). The heat denaturation of the RNA-proflavine complexes under different ionic conditions has been also investigated. The melting curves for double-stranded RNA-proflavine complex had two waves at high and low ionic strengths. For the single-stranded RNA the high temperature wave occured only at the high ionic strength. The dependence of heat denaturation of single-stranded RNA on ionic strength was examined for the evaluation of RNA structure. In these experiments a significant decrease of the width of melting interval under low ionic strength conditions was observed. It may reflect the existance in the RNA molecule of long helical regions. The occurence of such structures is likely to be responsible for an increase in the number of binding sites at low ionic strength and also for the appearance of the second wave on the melting curves of the single-stranded RNA-proflavine complexes at high ionic strength.

Acridines↗
PubMed1992

Unusual structure of the human immunodeficiency virus type 1 trans-activation response element.

The trans-activation response element (TAR) of human immunodeficiency virus type 1 is a structured RNA consisting of the first 60 nucleotides of all human immunodeficiency virus type 1 RNAs. Computer analyses and limited structural analyses indicated that TAR consists of a stem-bulge-loop structure. Mutational analyses showed that sequences in the bulge are required for Tat binding, whereas sequences in both the bulge and the loop are required for trans activation. In this study, we probed the structures of TAR and various mutants of TAR with chemical probes and RNases and used these methods to footprint a Tat peptide on TAR. Our data show that the structure of wild-type TAR is different from previously published models. The bulge, a Tat-binding site, consists of four nucleotides. The loop is structured, rather than simply single stranded, in a fashion reminiscent of the structures of the tetraloop 5'-UUCG-3' and the GNRA loop (C. Cheong, G. Varani, and I. Tinoco, Jr., Nature [London] 346:680-682, 1990; H.A. Heus and A. Pardi, Science 253:191-193, 1991). RNA footprint data indicate that three bases in the bulge are protected and suggest that a conformational change occurs upon Tat binding.

Base Sequence↗
PubMed1996

Translational recoding induced by G-rich mRNA sequences that form unusual structures.

We investigated a herpesvirus mutant that contains a single base insertion in its thymidine kinase (tk) gene yet expresses low levels of TK via a net +1 translational recoding event. Within this mutant gene, we defined a G-rich signal that is sufficient to induce recoding. Unlike other translational recoding events, downstream RNA structures or termination codons did not stimulate recoding, and paused ribosomes were not detected. Mutational analysis indicated that specific tRNAs or codon-anticodon slippage were unlikely to account for recoding. Rather, recoding efficiency correlated with the G-richness of the signal and its ability to form unusual structures. These findings identify a mechanism of translational recoding with unique features and potential implications for clinical drug resistance and other biological systems.

Amino Acid Sequence↗
PubMed1984

Synthesis and conformational studies of ribooligonucleotides which contain an alternating C-G sequence and show unusual circular dichroism spectra.

The poly [r(C-G)] duplex shows an unusually large negative CD band in the long wavelength region. In order to elucidate this phenomenon, r(C-G-C-G) and r(C-G-C-G-C-G) were synthesized by a phosphotriester method and their properties were examined by UV, CD, 1H and 31P NMR spectroscopy. These ribooligomers form self-duplexes at low temperature, the CD spectra of which show negative bands at around 290 nm and positive bands at around 265 nm. The results of 1H nuclear Overhauser effect experiments, 1H chemical shift-temperature profiles of base protons, and the sharp singlet observed for all H1' protons are consistent with a normal A-RNA structure but not with a Z-DNA like structure. The CD-temperature profiles and 31P NMR spectra support this conclusion. These results indicate that RNA duplexes with an alternating C-G sequence can give an unusually large negative CD band in the long wavelength region despite their right-handed helical structure.

Base Sequence↗
PubMed1996

Arginine-binding RNAs resembling TAR identified by in vitro selection.

Specific binding of the human immunodeficiency virus Tat protein to its RNA site (TAR) is mediated largely by a single arginine residue located within a basic region of the protein. Many essential features of the interaction can be mimicked by the free amino acid arginine, and an NMR model has been proposed in which the arginine guanidinium group binds to a guanine base in the major groove and to two phosphates adjacent to a bulge, with the RNA structure stabilized by a base triple between a U in the bulge and an adjacent A:U base pair. To compare the TAR structure to other arginine-binding RNAs, we performed in vitro selection experiments and identified RNAs with arginine-binding affinities similar to TAR. About 40% of the selected RNAs contained the same motif found in TAR: two stems separated by a bulge of at least two nucleotides, a U at the 5' position of the bulge, and G:C and A:U base pairs above the bulge. In many cases, the upper stems contained only the G:C and A:U pairs, located next to small loops. Chemical modification experiments demonstrated that these "TAR-like" RNAs bound arginine in a manner similar to TAR, and in some cases identified nucleotides outside the binding site that contributed to binding. To explore how small loops might help stabilize the structures of adjacent arginine-binding sites, we measured arginine-binding affinities of TAR-like RNAs having all possible three-nucleotide loops. An RNA with a UAG loop bound with highest affinity, and chemical modification and RNase mapping experiments suggested that the RNA changes conformation upon arginine binding, converting a large unstructured loop into a bulge conformation related to that of TAR. The results suggest that the arginine-binding site in TAR is structurally versatile and demonstrate how binding can be modulated by the surrounding RNA context.

Arginine↗
PubMed2005

Phenotypic analysis of human immunodeficiency virus type 1 Rev trimerization-interface mutants in human cells.

Nuclear export of unspliced and incompletely spliced human immunodeficiency virus type 1 mRNA is mediated by the viral Rev protein. Rev binds to a structured RNA motif known as the Rev-response element (RRE), which is present in all Rev-dependent transcripts, and thereby promotes entry of the ribonucleoprotein complex into the nuclear-export pathway. Recent evidence indicates that a dimerization interface and a genetically separable "trimerization" interface are required for multimeric assembly of Rev on the RRE. In this report, the effect of mutations within the trimerization interface on Rev function was examined in mammalian cells. All trimerization-defective Rev molecules had profoundly compromised Rev function and a range of localization defects was observed. However, despite the potential for formation of heterodimers between functional and non-functional Rev proteins, trimerization-defective Rev mutants were unable to inhibit wild-type Rev function in a trans-dominant-negative manner.

Active Transport, Cell Nucleus↗
PubMed1998

Functional requirements for specific ligand recognition by a biotin-binding RNA pseudoknot.

Ligand-binding RNAs and DNAs (aptamers) isolated by in vitro selection from random sequence pools provide convenient model systems for understanding the basic relationships between RNA structure and function. We describe a series of experiments that define the functional requirements for an RNA motif that specifies high-affinity binding to the carboxylation cofactor biotin. A simple pseudoknot containing an adenosine-rich loop accounts for binding in all independently derived aptamers selected to bind biotin, suggesting that it alone represents a global optimum for recognition of this particular nonaromatic, electrostatically neutral ligand. In contrast to virtually all previously identified aptamers, unpaired nucleotides make up a small fraction of the binding motif. Instead, the identity of 14 nucleotides involved in base pairing is highly conserved among functional clones and their substitution by nonidentical base pairs significantly reduces or eliminates binding. Chemical probing is consistent with the predicted pseudoknot motif and indicates that relatively little change in structure accompanies ligand binding, a strong contrast with results for other aptamers. Competition experiments suggest that the aptamer recognizes all parts of the biotin ligand, including its thiophane ring and fatty acid tail. Two alternative modes of binding are suggested by a three-dimensional model of the pseudoknot, both of which entail significant interactions with base-paired nucleotides.

Base Pairing↗
PubMed1994

Identification of the caprine arthritis encephalitis virus Rev protein and its cis-acting Rev-responsive element.

Caprine Arthritis Encephalitis Virus (CAEV) is a lentivirus closely related to visna virus of sheep and more distantly related to the human lentivirus HIV-1. The genomes of lentiviruses contain additional genes that regulate the lentivirus gene expression; one of these is Rev, a protein that regulates the expression of viral proteins via post-transcriptional mechanisms. A cDNA clone was isolated from CAEV infected cells and shown to encode the 18-kDa Rev protein of CAEV. Antibodies against CAEV Rev (Rev-C) demonstrated that the CAEV Rev protein accumulated in the nucleus and in particular in the nucleolus of transiently transfected cells. Mutation of a basic region in the CAEV Rev protein resulted in loss of nucleolar localization. A highly structured RNA element has been identified in the env gene of CAEV (nt 7850-8150); its structure and location suggested that it was analogous to the Rev-responsive element (RRE) of HIV-1 and visna virus. A 300-bp fragment (nt 7850-8150) spanning this region was substituted for the HIV-1 RRE in an HIV-1 Gag expression vector. Expression of the Gag protein was dramatically increased when Rev-C was added in trans, indicating that this fragment contained the cis-acting CAEV Rev Responsive Element. Cross-activation by the Rev/Rex proteins of other lentiviruses and members of the HTLV-I family indicated that this RRE could interact with Rev or Rex proteins of other viruses. This suggests that the highly divergent lentiviruses share similar mechanisms and cofactors regulating post-transcriptional viral gene expression. The Rev/RRE mechanism is thus the most conserved regulatory mechanism in lentiviruses and other complex retroviruses.

Amino Acid Sequence↗
PubMed2001

The 3' stem loop of the West Nile virus genomic RNA can suppress translation of chimeric mRNAs.

Cis-acting elements that regulate translation have been identified in the 3' noncoding regions (NCRs) of cellular and viral mRNAs. As one means of analyzing the effect on translation of the conserved 3' terminal RNA structure of the West Nile virus (WNV) genome, the translation efficiencies of chimeric mRNAs composed of a CAT reporter gene flanked by viral or nonviral 5' and 3' terminal sequences were compared. In vitro, the WNV 3'(+) stem loop (SL) RNA reduced the translation efficiencies of chimeric mRNAs with either viral or nonviral 5' NCRs, suggesting that a specific 3'-5' RNA-RNA interaction was not involved. In contrast, the 3' terminal sequence of a togavirus, rubella virus, enhanced translation efficiency. The WNV 3'(+)SL reduced translation efficiency both in cis and in trans and of both capped and uncapped chimeric mRNAs. We have previously reported that three cellular proteins bind specifically to the WNV 3'(+)SL RNA. Competition between the WNV 3'(+)SL and the 5' terminus of the chimeric mRNAs for proteins involved in translation initiation could explain the translation inhibition observed.

Animals↗