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At least 883 records · Page 49Linked to original sources
PubMed2001

Specific metal-ion binding sites in a model of the P4-P6 triple-helical domain of a group I intron.

Divalent metal ions play a crucial role in RNA structure and catalysis. Phosphorothioate substitution and manganese rescue experiments can reveal phosphate oxygens interacting specifically with magnesium ions essential for structure and/or activity. In this study, phosphorothioate interference experiments in combination with structural sensitive circular dichroism spectroscopy have been used to probe molecular interactions underlying an important RNA structural motif. We have studied a synthetic model of the P4-P6 triple-helical domain in the bacteriophage T4 nrdB group I intron, which has a core sequence analogous to the Tetrahymena ribozyme. Rp and Sp sulfur substitutions were introduced into two adjacent nucleotides positioned at the 3' end of helix P6 (U452) and in the joining region J6/7 (U453). The effects of sulfur substitution on triple helix formation in the presence of different ratios of magnesium and manganese were studied by the use of difference circular dichroism spectroscopy. The results show that the pro-Sp oxygen of U452 acts as a ligand for a structurally important magnesium ion, whereas no such effect is seen for the pro-Rp oxygen of U452. The importance of the pro-Rp and pro-Sp oxygens of U453 is less clear, because addition of manganese could not significantly restore the triple-helical interactions within the isolated substituted model systems. The interpretation is that U453 is so sensitive to structural disturbance that any change at this position hinders the proper formation of the triple helix.

Animals↗
PubMed1988

Spectroscopic analysis of DNA base-pair opening by Escherichia coli RNA polymerase. Temperature and ionic strength effects.

The interaction of Escherichia coli RNA polymerase with poly[d(A-T)] and poly[d-(I-C)] was studied by difference absorption spectroscopy at temperatures, from 5 to 45 degrees C in the absence and presence of Mg2+. The effect of KCl concentration, at a fixed temperature, was studied from 12.5 to 400 mM. Difference absorption experiments permitted calculation of the extent of DNA opening induced by RNA polymerase and estimation of the equilibrium constant associated with the isomerization from a closed to an open RNA polymerase-DNA complex. delta H0 and delta S0 for the closed-to-open transition with poly[d(A-T)] or poly[d(I-C)] complexed with RNA polymerase are significantly lower than the values associated with the helix-to-coil transition for the free polynucleotides. For the RNA polymerase complexes with poly[d(A-T)] and poly[d(I-C)] in 50 mM KCl, delta H0 approximately 15-16 kcal/mol (63-67 kJ/mol) and delta S0 approximately 50-57 cal/K per mol (209-239 J/K per mol). The presence of Mg2+ does not change these parameters appreciably for the RNA polymerase-poly[d(A-T)] complex, but for the RNA polymerase-poly[d(I-C)] complex in the presence of Mg2+, the delta H0 and delta S0 values are larger and temperature-dependent, with delta H0 approximately 22 kcal/mol (92 kJ/mol) and delta S0 approximately 72 cal/K per mol (approx. 300 J/K per mol) at 25 degrees C, and delta Cp0 approximately 2 kcal/K per mol (approx. 8.3 kJ/K per mol). The circular dichroism (CD) changes observed for helix opening induced by RNA polymerase are qualitatively consistent with the thermally induced changes observed for the free polynucleotides, supporting the difference absorption method. The salt-dependent studies indicate that two monovalent cations are released upon helix opening. For poly[d(A-T)], the temperature-dependence of enzyme activity correlates well with the helix opening, implying this step to be the rate-determining step. In the case of poly[d(I-C)], the same is not true, and so the rate-determining step must be a process subsequent to helix opening.

Base Composition↗
PubMed1999

Circular antisense oligonucleotides inhibit growth of chronic myeloid leukemia cells.

BACKGROUND: Antisense represents a conceptually powerful method for regulating gene expression. However, antisense oligonucleotides developed to date manifest two serious limitations-nuclease susceptibility and nonspecific hybridization. Circular oligonucleotides may be superior to conventional linear oligonucleotides in both respects. First, circular agents, having no ends, are exonuclease-resistant. Second, they bind to complementary strands of RNA and DNA with a higher affinity than corresponding linear agents. METHODS AND RESULTS: We assessed the activity of circular phosphodiester deoxynucleotides using chronic myeloid cell lines by targeting polypurine sequences. To represent cells having a bcr3/abl2-type junction, we used K562 cells. A circle targeting a bcr polypurine sequence 385 nucleotides 5' to the junction decreased the cell number by day 5 with an IC(50) of 9 microM. To represent cells having a bcr2/abl2-type junction, we used BV173 cells. A circle targeting the bcr-abl junction itself decreased the cell number by day 7 with an IC(50) of 8 microM. Control oligonucleotides, whether the same sequence uncircularized or circles with the same nucleotide composition but in scrambled sequence, had little effect. Unlike linear agents, circles were stable when incubated in 10% serum. The amount of bcr-abl protein detected by Western blotting using a specific anti-bcr-abl antibody at 24 hr in antisense-treated BV173 cells was only 10% of that of cells treated with control circles, which demonstrates an antisense mechanism of action. CONCLUSIONS: Circular oligodeoxyribonucleotides (1) inhibit the accumulation of CML cells, (2) decrease the amount of bcr-abl protein per cell, (3) have sequence-selective activity, and (4) are more active than linear oligonucleotides containing only the base-pairing region.

Base Sequence↗
PubMed1997

Measurement of the frequency of histone displacement during the in vitro transcription of nucleosomes: RNA is a competitor for these histones.

Transcription through tandemly arranged nucleosomes was studied to determine the frequency at which the nucleosomes would disrupt and cause displacement of the associated histones to a competitor DNA. In order to more effectively preserve topological effects, the template that was used in the in vitro transcription system was a large covalently, closed circular plasmid (8.9 kb). The plasmid contained two promoters for T7 RNA polymerase, each separated by 4.4 kb, and transcription was done in the presence of topoisomerase I at physiological ionic strength. Nucleosome disruption was observed at an approximate frequency of 1 in 4 nucleosomes such that after several rounds of transcription on the plasmid 80% of the nucleosomes were disrupted. Unexpectedly, all four histones were found associated with the RNA rather than the competitor DNA. The histones bound the competitor DNA only after removal of the RNA by RNase A treatment. By analyzing the topological state of the competitor DNA, it was observed that the majority of the histones that were displaced from the RNA were able to re-form nucleosomes. Additional experiments were done to determine the reasons for the preferential binding of histones to the newly synthesized RNA. It was found that the large molecular weight RNA binds histones with an approximate 100-fold greater affinity relative to DNA when at physiological ionic strength. Within the cell, this high-affinity binding would be expected to require cellular mechanisms to regulate the interaction of RNA with histones. The relatively high frequency of displacement of all four histones during transcription is higher than what is observed in vivo and suggests that additional factors are needed to regulate this displacement. These observations are discussed and compared with previous studies that have examined the process of transcription through nucleosomes.

Animals↗
PubMed2005

The AUUCU repeats responsible for spinocerebellar ataxia type 10 form unusual RNA hairpins.

Spinocerebellar ataxia type 10 is an autosomal dominant disorder caused by expansion of a pentameric repeat tract (ATTCT.AGAAT)(n) in intron 9 of the gene that encodes ataxin-10. We have analyzed duplex DNA containing the repeat, the individual DNA strands, and the RNA that would be generated by transcription of the repeat. Circular dichroism and UV absorbance measurements suggest that the previously reported tendency of the repeat to unpair when supercoiled is probably related simply to GC content rather than reflecting any unusual property of the duplex. DNA containing d(ATTCT)9 forms a folded structure at relatively low temperatures, whereas the antisense strand, d(AGAAT)9, does not form a structure even at 0 degrees C. In contrast r(AUUCU)9 forms a folded structure under physiologically reasonable conditions. S1 nuclease analysis reveals a single region of hypersensitivity in the middle of the repeat tract, whereas V1 digestion is consistent with a hydrogen bonded or well stacked structure. CD spectroscopy shows that the structure is unimolecular and hydrogen bonded and has a significant amount of A-form helix. NMR spectroscopy demonstrates that these hydrogen bonds comprise an equal number of A.U and U.U base pairs. Our data thus suggest that the repeat forms an unusual RNA hairpin. Thus the ability to form an RNA hairpin seems to be a common property of those Repeat Expansion Diseases that are not recessively inherited and are caused by repeats that are transcribed but not translated.

Amino Acid Sequence↗
PubMed1990

The self-splicing RNA of Tetrahymena is trapped in a less active conformation by gel purification.

When the circular form of the self-splicing intervening sequence of Tetrahymena thermophila was purified by denaturing polyacrylamide gel electrophoresis by standard methods, the rate of its reaction with tetrauridylate decreased 150-fold at 30 degrees C and at least 1000-fold at 0 degrees C. The activity of the self-splicing RNA was restored by heating it to high temperature and letting it renature in the presence of Mg2+. The rate of reaction of tetrauridylate with the self-splicing RNA flanked by exons was also greatly decreased by gel purification. The difference in activation energies for the reaction of native and denatured intervening sequences suggests that a substantial conformational rearrangement of the gel-purified RNA occurs prior to reaction.

Animals↗
PubMed1992

Entamoeba histolytica extrachromosomal circular ribosomal DNA: analysis of clonal variation in a hypervariable region.

The ribosomal RNA genes of the protozoan parasite Entamoeba histolytica are highly repeated and display restriction fragment length polymorphism. Using a set of four DNA probes spanning the coding region and part of the flanking region of the E. histolytica ribosomal RNA genes, an analysis of the DNA bands generated by EcoRI digestion of Entamoeba DNA is presented. This analysis included five strains of E. histolytica, four strains of E. moshkovskii, and one strain each of E. invadens and E. terrapinae. No common bands were observed between E. histolytica and the other Entamoeba. Within E. histolytica, two bands were conserved in all strains while the others were polymorphic. Detailed analysis of DNA from independently isolated clones of the strain HM-1:IMSS of E. histolytica showed two bands to be highly polymorphic. Of these, the 4.4-kb band of clone 6 was further analyzed. Polymorphism in this band could even be demonstrated in cells of the same clone. Restriction enzyme analysis of this DNA band from two clones of HM-1:IMSS showed that the polymorphism may be due to variable numbers of DraI repeat units present in this DNA stretch.

Animals↗
PubMed1994

Senescence-specific mitochondrial DNA molecules in P. anserina: evidence for transcription and normal processing of the RNA.

In Podospora anserina the phenomenon of senescence was previously shown to be correlated with the presence of senescence-specific circular DNAs (senDNAs), resulting from the amplification of distinct regions (alpha, beta, gamma and epsilon) of the mitochondrial chromosome. The beta region gives rise to senDNAs with variable sizes, but sharing a 1-kb common sequence. Here, we present a molecular analysis of five beta senDNAs. We have determined the nucleotide sequence around the circularization site of each senDNA monomer. In two cases, the presence of a tRNA gene, very close to the 3' end of the monomer, has been observed. This suggests that some beta senDNAs could be generated via a reverse transcription step. We have furthermore shown that the beta senDNAs produce specific transcripts which undergo normal processing of their introns. We propose that a transcription start site, located in the beta common region, is involved in mitochondrial replication allowing the amplification of the beta senDNAs.

Base Sequence↗
PubMed2000

Redundant and non-functional guide RNA genes in Trypanosoma brucei are a consequence of multiple genes per minicircle.

The mitochondrial mRNA of the parasitic protozoa Trypanosoma brucei is extensively edited by the insertion, and occasional deletion, of uridine residues. The editing is mediated by over 200 guide RNAs (gRNAs) that are encoded in circular DNA molecules called minicircles. There are some 250 different types of minicircle, called classes, with each encoding several gRNAs. Sequencing of gRNAs and minicircles has revealed a surprising amount of both redundancy, where gRNAs from different minicircle classes edit exactly the same part of an mRNA, and non-functionality, where partial or no complementarity is found between gRNA and mRNA. How does this redundancy and non-functionality arise and persist? We propose the following. Minicircle classes that contain several functional gRNA genes can be lost from the population via drift and replaced by more minicircle classes that contain fewer functional gRNA genes, on the condition that the cells keep a full complement of functional gRNAs. We demonstrate this hypothesis in a computer simulation of a model of minicircle evolution. We show that this process leads to an increasing number of minicircle classes and inevitably to only one functional gRNA per minicircle. Moreover, we show that the genome contains more minicircle classes than is actually necessary for cell survival. We also analyse the available minicircle sequence data and conclude that T. brucei is at a transient stage in this process. In addition, ten new putative gRNAs have been discovered.

Animals↗
PubMed1976

Influence of DNA acidification on DNA premelting and template properties.

Acidification of a T7 DNA sample was found to be partly irreversible as ultraviolet difference spectra measured at various sub-melting temperatures were different from those observed for a 'normal' DNA sample. This implies some subtle conformational change which is not reversed by return to neutral pH. In the same conditions, only poly(purine)-poly(pyrimidine) polymers behaved in a different manner, during premelting, according to whether they were previously acidified or not. The properties of acidified and reneutralized T7 DNA were also investigated for Escherichia coli RNA polymerase binding and transcription. An inhibition of RNA synthesis and chain initiation was observed. The results suggest that the binding of the enzyme is affected. RNA synthesized is specific but there is a decrease in the number and in the stability of the RNA-polymerase-DNA complexes.

Binding Sites↗
PubMed1993

Ribonucleoprotein complexes of hepatitis delta virus.

Human hepatitis delta virus (HDV) is a subviral satellite agent of hepatitis B virus (HBV). The envelope proteins of HDV are provided by the helper virus, HBV, but very little is known about the internal structure of HDV. The particles contain multiple copies of the delta antigen and an unusual RNA genome that is small, about 1,700 nucleotides in length, single stranded, and circular. By using UV cross-linking, equilibrium density centrifugation, and immunoprecipitation, we obtained evidence consistent with the interpretation that delta antigen and genomic RNA form a stable ribonucleoprotein (RNP) complex within the virion. Furthermore, electron-microscopic examination of the purified viral RNP revealed a roughly spherical core-like structure with a diameter of 18.7 +/- 2.5 nm. We also isolated HDV-specific RNP structures from the nuclei of cells undergoing HDV genome replication; both the genome and antigenome (a complement of the genome) of HDV were found to be in such complexes. From the equilibrium density analyses of the viral and nuclear RNPs, we were able to deduce the number of molecules of delta antigen per molecule of HDV RNA. For virions, this number was predominantly ca. 70, which was larger than for the nuclear RNPs, which were more heterogeneous, with an average value of ca. 30.

Animals↗
PubMed1990

A site-specific self-cleavage reaction performed by a novel RNA in Neurospora mitochondria.

We describe a novel DNA and RNA found in the mitochondria of the Varkud-1c natural isolate of Neurospora. The majority of the RNA, termed VSRNA, is an 881 nucleotide single-stranded circular molecule complementary to one strand of a low copy, double-stranded circular DNA, VSDNA. VSRNA combines some features of the human hepatitis delta virus, group I introns, retroelements, and plant viral satellite RNAs. VSRNA synthesized in vitro performs a self-cleavage reaction whose products terminate with a 5' hydroxyl and a 2',3' cyclic phosphate. This reaction may be involved in the natural processing pathway of multimeric VSRNA in vivo. VSRNA lacks a hammer-head structure or substantial sequence similarity to any other self-cleaving RNA, suggesting that the RNA structure involved in cleavage may be different from those in previously characterized catalytic RNAs.

Base Sequence↗
PubMed1983

Mechanism of stimulation by a specific protein factor of de novo DNA synthesis by mouse DNA replicase with fd phage single stranded circular DNA.

Mouse DNA replicase is a functional multienzyme complex consisting of DNA polymerase and DNA primase. The DNA and initiator RNA syntheses by DNA replicase with single stranded DNA as template are stimulated by a stimulating factor (T. Yagura, T. Kozu and T. Seno, 1982, J. Biochem. (Tokyo).91, 607-618). The action mechanism of the stimulating factor on this novel DNA synthesis with fd phage single stranded circular DNA as template was studied. The stimulating factor directly stimulated initiator RNA synthesis but did not change the length of either initiator RNA (8 to 10 nucleotides long) or the product DNA (300 to 1,000 nucleotides long). Kinetic studies and analysis of the products by neutral agarose gel electrophoresis show that the stimulating factor increased the affinity of DNA replicase for template DNA without changing the apparent Km values for deoxy- and ribonucleotide substrates. Thus, in combination with a sufficient amount of the stimulating factor, DNA replicase quantitatively converted the template DNA to the position of double-stranded circular replicative form II DNA, as shown by agarose gel electrophoresis.

Animals↗
PubMedDate not supplied

[Characteristics of the structural organization and transcriptase activity of the influenza virus nucleoid].

The state of the secondary structure of RNA and proteins comprising nucleoids (cores) and RNP of influenza virus was evaluated comparatively. The identity of RNA conformation in these particles and differences from free RNA conformation due to less marked secondary structure were found. Core proteins were predominantly represented by the beta-framework, and RNP as an alpha-helix. The specific transcriptase activity of the core is significantly lower than RNP activity of influenza virus.

Circular Dichroism↗
PubMed1985

Specific fragmentation of tRNA and rRNA at a 7-methylguanine residue in the presence of methylated carrier RNA.

The reaction of site-specific cleavage of tRNA at a 7-methylguanine residue, including subsequent treatment with sodium borohydride and aniline [Wintermeyer, W. and Zachau, H.G. (1975) FEBS Lett. 58, 306-309], was shown to work only within a certain range of tRNA concentrations (higher than 30 microM). The Escherichia coli 16S rRNA, which contained a unique m7G (position 527), could not be split by this method when taken at any concentration. It was found that the presence of statistically methylated carrier RNA in the reaction mixture at the borohydride stage significantly stimulates site-specific fragmentation of 16S rRNA and 32P-labeled tRNAs. Direct sequencing proved that 16S rRNA and tRNA are cleaved by this procedure successfully at the m7G residue. The E. coli 16S rRNA was preparatively cleaved by the described procedure into two fragments. The 5'-terminal fragment (1-526) and the 3'-terminal fragment (528-1542) were isolated in the pure form and their secondary structure investigated by the circular dichroism method. The results of this study showed that the secondary and tertiary structures of the 5'-terminal one-third of the 16S rRNA are at least as ordered as those of intact 16S rRNA or its 3'-terminal two-thirds.

Base Sequence↗
PubMed1994

Costabilization of peptide and RNA structure in an HIV Rev peptide-RRE complex.

An arginine-rich peptide corresponding to amino acids 34-50 of the human immunodeficiency virus Rev protein has been shown to bind specifically to its RNA-binding site (RRE) when the peptide is in an alpha-helical conformation. Mutation of any one of six amino acids (Thr34, Arg35, Arg38, Arg39, Asn40, or Arg44) was shown to strongly decrease specific RNA-binding affinity in vitro, suggesting that these residues may contact specific bases or distinct structural features of the RNA. We now show that the four arginine side chains, and not just their charge, are important for specific binding in vivo, and present evidence that three additional arginines (Arg46, Arg48, and Arg50) may make electrostatic contacts to the RRE. RNA-binding specificity of the Rev peptide is temperature-dependent in vitro, correlating with alpha-helix unfolding. Circular dichroism experiments indicate that the peptide helical structure is stabilized when bound specifically to the RRE and that the RNA undergoes a conformational change upon binding. Because the structures of the peptide and RNA in this model system appear to be mutually stabilized upon binding, it is suggested that the entire complex may be viewed as a single folding unit.

Amino Acid Sequence↗
PubMed1989

Self-cleavage of RNA in the replication of small pathogens of plants and animals.

The ability of certain small, circular, pathogenic RNAs of plants and animals to self-cleave at specific sites in vitro in the complete absence of protein most likely plays a central role in their replication in vivo by a rolling circle mechanism. The self-cleavage of an RNA transcript from a satellite DNA of the newt indicates that this reaction is not limited to pathogenic RNAs. Further, the site-specific self-cleavage in trans by two separate RNA molecules suggests that such reactions may be important in gene regulation in normal cells as well as in the genesis of symptom expression on infection by RNA pathogens.

Animals↗