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Results for “Circular RNA”

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At least 469 records · Page 26Linked to original sources

PubMed1991

Evidence for spontaneous circle formation in the replication of the satellite RNA of tobacco ringspot virus.

Replication of the satellite RNA of tobacco ringspot virus (sTobRV RNA) has been postulated to require rolling circle transcription. The expected product of rolling circle transcription, multimeric sTobRV RNA, is known to undergo self-cleavage in vitro to release unit-length sTobRV RNA. A spontaneous, efficient, not enzymically-catalyzed in vitro circularization reaction is characteristic of unit-length sTobRV RNA of the less abundant, (-) polarity. We mutated sTobRV RNA at two sites that are distant from each other in the polyribonucleotide chain. A third form of the sTobRV RNA was mutated at both sites. Multimeric forms of the one-site mutants of sTobRV(+)RNA and sTobRV(-)RNA showed, respectively, undiminished and slightly diminished self-cleavage, whereas the spontaneous circularization of each one-site-mutated, unit-length sTobRV(-)RNA was greatly reduced, compared to the reactions of wild-type sTobRV RNA and the two-site mutant. The two-site mutant and the wild-type sTobRV RNAs replicated with similar efficiency. They reduced the titer of, and severity of, symptoms induced by coinoculated tobacco ringspot virus (TobRV). When coinoculated with TobRV, neither one-site mutant increased or provided protection against TobRV. Rather, each induced a substantial accumulation of what is apparently an endogenous form of sTobRV RNA. Our results are consistent with the formation of circular sTobRV(-)RNA as an essential step in sTobRV RNA replication.

Base Sequence↗

PubMed2006

RNA and CuCl2 induced conformational changes of the recombinant ovine prion protein.

Prion diseases are a group of neurodegenerative illnesses caused by conformational conversion of benign, alpha-helix rich cellular prion protein (PrP(C)) into the highly stable, beta-sheet rich scrapie prion protein (PrP(Sc)) isoform. To date, the role of RNA on the conformational conversion of ovine prion protein in vitro remains unknown. To examine the effect of the interaction between RNA and PrP(C), conformations of recombinant ovine prion protein PrP23-256 (OvPrP23-256) binding various concentrations of RNA were analyzed by circular dichroism (CD) spectrum. The results indicated that the conformational conversion of OvPrP23-256 was triggered by RNA with a decrease in alpha-helix content and increase in beta-sheet. Moreover, the conformation of OvPrP23-256 interacting with both RNA and CuCl2 was also examined by CD spectrum, which showed that alpha-helix content decreased while beta-sheet increased dramatically. Proteinase K digestion assay disclosed that the recombinant ovine PrP(C) acquired PK resistance after RNA and/or Cu2+ treatment. It confirmed that the RNA/Cu2+ treatment in vitro altered the biochemical properties of ovine PrP(C). The implication of this finding, with respect to PrP(Sc), is that a dysfunctional state of a normal physiological process possibly facilitates diseases. The information gained from this study may provide useful approaches to study the pathogenesis of prion diseases.

Animals↗

PubMed1978

Transcription and circular dichroism of chromatin in lymphoblastoid cell lines during proliferation and quiescence.

RNA polymerase activity of nuclei and positive circular dichroism ellipticity in the 250--300 nm region of chromatin were studied in three lymphoblastoid cell lines: HRI, an Epstein-Barr Virus (EBV) positive virus-producing line; Raji, and EBV positive virus-producing line; Raji, an EBV positive non-producer line; and SU-AMB-1, and EBV negative line. Raji consistently exhibited the highest and SU-AMB-1 the lowest polymerase activity and ellipticity. All lines showed higher RNA polymerase activity and positive ellipticity when exponentially growing than when stationary. In Raji it was shown that changes in RNA polymerase activity and chromatin ellipiticity were parallel. The changes in RNA polymerase activity of nuclei and circular dichroism ellipticity correlated with those of the proliferative state but not with the number of viral genomes or the production of virus or viral antigens.

Cell Division↗

PubMed1975

Circular DNA and rolling circles in nucleolar rDNA from mitotic nuclei of Physarum polycephalum.

1. About 15% of nucleolar DNA (1.712 g/cm3) from Physarum polycephalum displaying maximum hybridization to ribosomal RNA, is composed of circular DNA of 3.9 +/- 0.2 mum contour length or multiples thereof. 2. A portion of these circular molecules (25%) contained linear DNA pieces longer than circumference length. In a small fraction of circular DNA linear pieces, shorter than the unit length, were observed. 3. Most nucleolar DNA, [3H]thymidine-labeled or hybridizable to ribosomal RNA was separable from chromosomal DNA during G2 phase, mitosis and S phase of the cell cycle. 4. Ribosomal DNA content was not amplified during the cell cycle, was unchanged during exponential or stationary growth phase and amounted to about 0.11 -- 0.21% of nuclear DNA in diploid and hexaploid strains of Physarum or 100--200 ribosomal genes per diploid genome.

Cell Division↗

PubMed2004

Reduced contact order and RNA folding rates.

We investigated the relationship between RNA structure and folding rates accounting for hierarchical structural formation. Folding rates of two-state folding proteins correlate well with relative contact order, a quantitative measure of the number and sequence distance between tertiary contacts. These proteins do not form stable structures prior to the rate-limiting step. In contrast, most secondary structures are stably formed prior to the rate-limiting step in RNA folding. Accordingly, we introduce "reduced contact order", a metric that reflects only the number of residues available to participate in the conformational search after the formation of secondary structure. Plotting the folding rates and the reduced contact order from ten different RNAs suggests that RNA folding can be divided into two classes. To examine this division, folding rates of circularly permutated isomers are compared for two RNAs, one from each class. Folding rates vary by tenfold for circularly permuted Bacillus subtilis RNase P RNA isomers, whereas folding rates vary by only 1.2-fold for circularly permuted catalytic domains. This difference is likely related to the dissimilar natures of their rate-limiting steps.

Bacillus subtilis↗

PubMed1994

Detection of circular and linear transcripts of Sry in pre-implantation mouse embryos: differences in requirement for reverse transcriptase.

Sry is the testes determining factor gene located on the Y chromosome. Expression of Sry had previously been found to occur during a short time frame of 10.5 to 12.5 days post coitum in the developing genital ridge. A recent study of the Sry transcript from adult testes found that the most abundant transcript is circular in nature. We have performed reverse transcription polymerase chain reactions with RNA from mouse preimplantation embryos ranging from the 2 cell stage to the blastocyst stage and report the detection of both circular and linear forms of Sry in the preimplantation embryo. In addition, we also demonstrate that the well described reverse transcriptase activity of Taq polymerase leads to a major difference in the mode of detection of the two forms.

Animals↗

PubMed1975

The genome of RNA tumor viruses: a functional requirement for a polyploid structure?

This paper points out certain theoretical problems in DNA synthesis associated with antiprimer transcription and with circularization that could oblige RNA tumor viruses to rely on a polyploid genome. It is suggested that each completed act of reverse transcription may be coupled with an act of genetic recombination aimed at recovering the antiprimer information from an adjacent genome subunit in a polyploid train. A partially double-stranded DNA transcript could then be formed with sufficient terminal redundancy to permit circularization. The model provides satisfactory explanations for observed genetic interactions (particularly recombination and heterozygote formation), for inactivation data and for selective subunit transcription.

DNA, Viral↗

PubMed1979

Identification and mapping of the transcriptional and translational products of the yeast plasmid, 2mu circle.

We have identified two major and approximately ten minor poly(A)-containing RNA species in S. cerevisiae which arise from in vivo transcription of the yeast plasmid, known as 2mu circle. The two major species, which are 1325 and 1275 bases in length, are transcribed from the two unique halves of the plasmid and extend into the inverted repeat sequences which separate the unique regions. The map positions of the minor transcripts, which range in length from 350 to 2600 bases, indicate that except for a small region of the genome in which no transcription is observed, both strands of the entire 2mu circle genome are transcribed. We also present evidence demonstrating that RNA transcribed from 2mu circular DNA is used to program the synthesis of specific proteins in yeast: that is, yeast RNA complementary to 2mu circle DNA can be translated in vitro to produce specific polypeptides of substantial size. Finally, the pattern of transcription of 2mu circle suggests the possibility that messenger RNA species are derived by cleavage of larger transcripts, and in addition, that the intramolecular recombination of 2mu circle which occurs in yeast functions as a genetic switch to allow separate expression of two sets of genes on the 2mu circle genome.

Binding Sites↗

PubMed1995

Only one of four possible secondary structures of the central conserved region of potato spindle tuber viroid is a substrate for processing in a potato nuclear extract.

The influence of RNA secondary structure on the substrate activity of a longer-than-unit length transcript for processing to circular viroids was studied in a nuclear extract from potato suspension cells. The nuclear extract was prepared according to a modified procedure for a plant transcription extract. The transcript of the potato spindle tuber viroid (PSTVd) consists of a monomeric molecule with 17 additional nucleotides, thus doubling most of the central conserved region of viroids of the PSTVd-class. The transcript can assume four different secondary structures, which either co-exist as conformers in solution or can be kept as metastable structures after different treatments by temperature and/or ionic strength. The structures were analysed by thermodynamic calculations and temperature-gradient gel electrophoresis and were confirmed by oligonucleotide mapping. Only the so-called extended middle structure was processed to exact viroid circles. In this structure the 5'- and 3'-ends are branching out from the rod-like viroid structure at the loop starting with nucleotide 87. The other structures were processed only if they could be rearranged into the active structure.

Base Sequence↗

PubMed1982

Photoreactivating enzyme from Escherichia coli: isolated enzyme lacks absorption in its actinic wavelength region and its ribonucleic acid cofactor is partially double stranded when associated with apoprotein.

Isolated photoreactivating enzyme (PRE) from Escherichia coli exhibits some optical density at wavelengths greater than 300 nm. After correcting for the effects of light scattering, however, we find no true absorption in the spectral region that is required for enzymatic activity (320-450 nm). At shorter wavelengths, there is an absorption maximum near 260 nm that is due primarily to an RNA cofactor. Heating to 60 degrees C and subsequently cooling to 4 degrees C release the RNA cofactor from association with apoprotein and result in hyperchromicity. Circular dichroism indicates that the RNA associated with native enzyme is partially double stranded. At low ionic strength (mu = 0.01), heating to 15 degrees C or protease treatment at 4 degrees C results in irreversible loss of part of the double strandedness. We show that the difference spectrum at 4 degrees C between the absorption spectra of native enzyme and heat-treated enzyme can be fit by a superposition of reference spectra for denaturation of A-U and G-C base pairs derived from model polynucleotides. The coefficients of the linear combination of reference spectra were used to calculate the fraction of A-U and G-C base pairs. We find that both A-U and G-C base pairs are present in equal concentrations and that about 20% are in a double-stranded conformation in the native enzyme.

Base Composition↗

PubMed2003

A spectrophotometric method to quantify linear DNA.

A spectrophotometric method for quantification of linear DNA is described. The assay measures ADP produced following digestion of linear DNA by an ATP-dependent deoxyribonuclease. Cleavage of the phosphodiester bond of the DNA substrate is proportional to ADP formed in the reaction which follows typical Michaelis-Menten kinetics (K(m) of 0.6 microM, and a V(max) of 30 nmol/min/mg). The enzyme requires Mg(2+)-ATP and Mg(2+)-DNA as substrates, although the results suggest a requirement for yet another metal ion which may be enzyme bound. Both single-stranded and double-stranded linear DNA are substrates, as demonstrated by comparable initial velocity measurements. However, covalently closed circular (CCC) and nicked open circular DNA are not substrates for the enzyme. The rate of hydrolysis of ATP is not inhibited by 1 microg RNA or covalently closed circular DNA. The product (ADP) formed in the reaction is coupled to NADH oxidation using pyruvate kinase and lactate dehydrogenase. NAD formed in the reaction is monitored spectrophotometrically as a loss in absorbance at 340 nm. This assay directly measures the amount of linear DNA present in preparations of supercoiled (CCC) plasmid DNA, and has direct utility for monitoring the quality of plasmid preparations for gene therapy.

Adenosine Diphosphate↗

PubMed2005

Regulation of the structure of RNA assemblies through RNA tertiary interactions.

We have previously shown that RNA building blocks containing two or three stem-loops based on the dimerization initiation site (DIS) connected by short linkers can be assembled into various one- and two-dimensional structures. Here, in an attempt to regulate the conformation of the RNA assemblies by adjusting the rigidity of the RNA building block, we have introduced the triple-helical scaffold (THS) into the linker region connecting two DIS-based stem-loops. As a result, linear and circular assemblies could be selectively formed with RNA building blocks containing the wild-type and mutant THS sequences, respectively. The results demonstrate how the conformation of RNA assemblies may be regulated by external factors, thereby expanding the utility of RNA-based nanostructures.

Dimerization↗

PubMed1992

Influence of chromatin folding on transcription initiation and elongation by RNA polymerase III.

Nucleosomes were assembled onto either closed circular plasmids containing a single Xenopus 5S RNA gene or a linear tandemly repeated array of Lytechinus 5S RNA genes. Both chromatin templates were found to vary in their extent of compaction, depending upon the type and concentration of cation in solution. Compaction of these chromatin templates led to a significant inhibition of both transcription initiation and elongation by RNA polymerase III. Thus, the transcriptional repression observed after incorporation of genes into chromatin depends not only on occlusion of the promoter elements through direct contact with histones but also on compaction of nucleosomal arrays which occurs under the conditions of the transcription reactions.

Animals↗

PubMed1998

Analysis of the conformation of the 3' major domain of Escherichia coli16S ribosomal RNA using site-directed photoaffinity crosslinking.

The 3' major domain of Escherichia coli 16S rRNA, which occupies the head of the small ribosomal subunit, is involved in several functions of the ribosome. We have used a site-specific crosslinking procedure to gain further insights into the higher-order structure of this domain. Circularly permuted RNAs were used to introduce an azidophenacyl group at specific positions within the 3' major domain. Crosslinks were generated in a high-ionic strength buffer that has been used for ribosome reconstitution studies and so enables the RNA to adopt a structure recognized by ribosomal proteins. The crosslinking sites were identified by primer extension and confirmed by assessing the mobility of the crosslinked RNA lariats in denaturing polyacrylamide gels. Eight crosslinks were characterized. Among them, one crosslink demonstrates that helix 28 is proximal to the top of helix 34, and two others show that the 1337 region, located in an internal loop at the junction of helices 29, 30, 41, and 42, is proximal to the center of helix 30 and to a segment connecting helix 28 to helix 29. These relationships of vicinity have previously been observed in native 30S subunits, which suggests that the free domain adopts a conformation similar to that within the 30S subunit. Furthermore, crosslinks were obtained in helix 34, which suggest that the upper and lower portions of this helix are in close proximity.

Azides↗

PubMed2003

The coat protein of prunus necrotic ringspot virus specifically binds to and regulates the conformation of its genomic RNA.

Binding of coat protein (CP) to the 3' nontranslated region (3'-NTR) of viral RNAs is a crucial requirement to establish the infection of Alfamo- and Ilarviruses. In vitro binding properties of the Prunus necrotic ringspot ilarvirus (PNRSV) CP to the 3'-NTR of its genomic RNA using purified E. coli- expressed CP and different synthetic peptides corresponding to a 26-residue sequence near the N-terminus were investigated by electrophoretic mobility shift assays. PNRSV CP bound to, at least, three different sites existing on the 3'-NTR. Moreover, the N-terminal region between amino acid residues 25 to 50 of the protein could function as an independent RNA-binding domain. Single exchange of some arginine residues by alanine eliminated the RNA-interaction capacity of the synthetic peptides, consistent with a crucial role for Arg residues common to many RNA-binding proteins possessing Arg-rich domains. Circular dichroism spectroscopy revealed that the RNA conformation is altered when amino-terminal CP peptides bind to the viral RNA. Finally, mutational analysis of the 3'-NTR suggested the presence of a pseudoknotted structure at this region on the PNRSV RNA that, when stabilized by the presence of Mg(2+), lost its capability to bind the coat protein. The existence of two mutually exclusive conformations for the 3'-NTR of PNRSV strongly suggests a similar regulatory mechanism at the 3'-NTR level in Alfamo- and Ilarvirus genera.

3' Untranslated Regions↗

PubMed1985

Rise per base pair in helices of double-stranded rotavirus RNA determined by electron microscopy.

Regular helices of double-stranded RNA occur in nature only as the genome of certain viruses. The structure of such double-stranded RNA helices has been little studied compared to that of DNA, but some X-ray crystallographic data (Arnott, 1970; Saenger, 1984) are available. The recent advent of sequence data of bovine rotavirus RNA (Dyall-Smith et al., 1983; Elleman et al., 1983; Ward et al., 1984) has enabled us to determine by direct measurement of electron micrographs the translation, or axial distance between base pairs in RNA duplexes. Using two different spreading conditions we obtained values of 2.79 +/- 0.10 and 2.80 +/- 0.11 A. These results are consistent with the 11-fold A RNA (Arnott, 1970; Rosenberg et al., 1976) proposed for the conformation of double-stranded RNA. We included both circular and linear molecules of phi X174 RF DNA in the same preparations, and the translations for these molecules were between 3.23 +/- 0.06 and 3.29 +/- 0.05 A. Thus, double-stranded RNA contained 1.16 to 1.17 times more nucleotides per unit length than DNA.

Animals↗

PubMed1998

Specific RNA self-cleavage in coconut cadang cadang viroid: potential for a role in rolling circle replication.

The rolling circle replication of the small, single-stranded viroid RNAs requires a specific processing reaction to produce monomeric RNAs that are ligated into the final circular form. For avocado sunblotch viroid, peach latent mosaic viroid, and chrysanthemum chlorotic mottle viroid, the hammerhead self-cleavage reaction is considered to provide this processing reaction. We have searched for a similar type of reaction in the 246-nt coconut cadang cadang viroid, the smallest viroid of the 24-member potato spindle tuber viroid (PSTV) group. RNA transcripts prepared from the cloned central or C domain of this viroid self-cleaved specifically after denaturation with methylmercuric hydroxide followed by incubation in the presence of spermidine but in the absence of added magnesium ions. The unique cleavage site was located in the bottom strand of the C domain within a potential hairpin structure that is conserved within members of all three subgroups of the PSTV group of viroids.

Base Sequence↗

PubMed1981

Binding of ribosomes to linear and circular forms of the 5'-terminal leader fragment of tobacco-mosaic-virus RNA.

The sequence of the 5'-terminal leader fragment preceding the AUG codon in the RNA of tobacco mosaic virus (TMV), tomato strain, SPS isolate, has been determined. This RNA, similarly to the RNAs of the U1 and Dahlemense strains of TMV [Kukla et al. (1979) Eur. J. Biochem. 98, 61--66] has the 7-methylguanosine(5')triphospho(5')guanosine cap separated from the initiation codon by a long stretch of nucleotides devoid of guanosine residues. The RNase-T1-resistant 73-nucleotide-long leader fragment of TMV RNA from the SPS isolate was assayed for its ability to interact with eukaryotic and prokaryotic ribosomes. The linear fragment, labelled either at its 5' or 3' end, efficiently formed disome initiation complexes when incubated with wheat-germ protein-synthesis extract. In contrast to its linear counterpart, the circular covalently closed RNA leader fragment, obtained in a reaction catalysed by T4 RNA ligase, was unable to interact with wheat germ ribosomes. Both kinds of leader fragment bound equally well to Escherichia coli 70-S ribosomes. The results offer further support to the notion that in eukaryotic initiation the free 5' end (either capped or uncapped) is required for mRNA interaction with ribosomes. Furthermore, they suggest that both ribosomes found in disome initiation complexes with the TMV RNA leader fragment enter the mRNA sequentially via the free 5' terminus.

Base Sequence↗