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At least 685 records · Page 38Linked to original sources
PubMed2003

Initial binding of the broad spectrum antiviral nucleoside ribavirin to the hepatitis C virus RNA polymerase.

Ribavirin is a broad spectrum antiviral nucleoside that displays activity against a variety of RNA and DNA viruses. Ribavirin is currently used in combination with interferon-alpha for the treatment of hepatitis C virus (HCV) infection and was recently shown to be directly incorporated by the HCV RNA polymerase into RNA products. This capacity ultimately leads to increased mutation rates and drastically reduces the viral fitness. As a first step toward elucidating the nature of the specific interaction between ribavirin and the HCV polymerase, we have utilized fluorescence spectroscopy to monitor precisely the binding of ribavirin triphosphate (RTP) to the viral polymerase. This spectroscopic approach allowed us to clearly separate the RTP binding activity from the concomitant catalytic steps. We report here the first detailed study of the binding kinetics and thermodynamic parameters involved in the interaction between RTP and an RNA polymerase. We demonstrate that RTP binds to the same active site as nucleotides. Furthermore, we provide evidence that the HCV polymerase cannot only bind to RTP but also to nonphosphorylated ribavirin, albeit with less affinity. By using various combinations of template-primers, we also demonstrate that base pairing is not involved in the initial binding of RTP to the HCV polymerase. Based on the results of circular dichroism and denaturation studies, we show that the RNA polymerase undergoes subtle conformational changes upon the binding of RTP, although the interaction does not significantly modify the stability of the protein. Finally, although metal ions are required for catalytic activity, they are not required for the initial binding of RTP to the polymerase. Such quantitative analyses are of primary importance for the rational design of new ribavirin analogues of potential therapeutic value and provide crucial insights on the interaction between RTP and the HCV RNA polymerase.

Adenosine Triphosphate↗
PubMed1976

Function of DNA polymerase I in RNA-primed synthesis of bacteriophage M-13 duplex DNA.

Cell-free extracts from Escherichia coli contain a DNA polymerase activity resistant to SH-blocking agents, which is capable of synthesizing complementary strand DNA on a circular M-13 DNA template by extension of RNA primers. This activity is considered to be identical with DNA polymerase I (or some altered form of this enzyme) since it is missing in extracts from po1A- cells. DNA synthesis in the presence of SH-blocking agents occurs at a reduced rate as compared to untreated controls and leads to the formation of DNA chains of defined size (0.4-0.5 genome's length). It is concluded that efficient M-13 duplex DNA synthesis requires the cooperation of both DNA polymerase I and III.

Chloromercuribenzoates↗
PubMedDate not supplied

[Circular dichroism study of the ribonucleoprotein structure of Sendai virus isolated from infected chick embryo cells].

Thermostability and secondary structure of RNA, a component of ribonucleoprotein (RNP) of Sendai virus isolated from infected chick embryo cells (cRNP) and from virions (vRNP) were studied by the method of circular dichroism. The secondary structure of RNA in cRNP was shown to be practically no different from that of free RNA whereas incorporation of RNA into vRNP was accompanied by a significant decrease in the number of paired bases in it. Comparison of thermodenaturation parameters of cRNP and vRNP also revealed significant differences in their structural organization. Thus, melting of cRNP as well as of free RNA is of markedly non-cooperative nature indicating poor RNA-protein interactions in the complex. In contrast, the process of vRNP thermodenaturation occurs in a step-wise manner in a narrow temperature range indicating a significant role in the maintenance of this RNP structure of both RNA-protein and, apparently, protein-protein interactions.

Animals↗
PubMed1980

Circular dichroism study of Escherichia coli initiation factor 3 binding to nucleic acids.

The circular dichroic spectral features of (A)10-20, (C)10-20, A8UGU6, poly(A), and poly(C), at both neutral and acidic pH values and in the presence and absence of Mg2+, are significantly altered by Escherichia coli initiation factor 3 (IF3), implying the occurrence of protein-induced changes in nucleic acid secondary structure. Similarly, the circular dichroic spectral characteristics of helical poly(U), poly-(A)-poly(U), and poly(I)-poly(C) are modified by IF3. However, no structural perturbation of poly(A)-poly(U) occurs in the absence of Mg2+ by IF3. The oligonucleotides (A)10-20 and (C)10-20 at both pH 7.5 and 5.5 titrate to end point of 26 +/- 4 nucleotide residues per IF3 [except (C) 10-20 at pH 5.5 which titrates to 17 +/- 1 nucleotide residues per IF3], whereas the hairpin A8UGU6 under similar conditions at neutral pH and in the presence of Mg2+ titrates to an end point of 56 +/- 3 nucleotide residues per IF3, thereby suggesting the presence of multiple binding sites on the protein. By contrast, poly(A) and poly(C) at neutral pH and in the absence of Mg2+ titrate to an end point of 13 +/- 1 nucleotide residues per IF3. The occurrence of significant light-scattering artifacts precluded a determination of the end point stoichiometry in most other cases. The circular dichroic spectra of E. coli tRNA, MS2 RNA, phiX174 DNA, and sonicated calf thymus DNA were unaffected by IF3 at physiological concentrations. Addition of an equimolar mixture of IF3 and ribosomal protein S1 titrates the circular dichroism of poly(C) at acid pH as did S1 alone. However, addition of IF3 to mixture of poly(A) and S1 at neutral pH did not result in significant titration of the optical activity until IF3 was in excess over S1, even though filter binding assays indicate normal IF3 binding to the polynucleotide. The possible relation of these observations to the biological function of IF3 is briefly considered.

Circular Dichroism↗
PubMed1994

A-Z-RNA conformational changes effected by high pressure.

This paper reports evidence obtained by circular dichroism (CD) spectroscopy measurements indicating that two oligoribonucleotide duplexes with the alternating purine-pyrimidine sequences r(GC)6 or r(AU)6 change their A-RNA conformation under high pressure. Under the high-pressure conditions at which B-Z-DNA transition easily occurs, RNA acquires a conformation which only differs slightly from that of A-RNA. However, exposure of r(GC)6 or r(AU)6 to high pressure (6 kbar) in the presence of 5 M NaCl causes a conformation change of both oligoribonucleotide duplexes from their A- to their Z-RNA form. The departure of RNA or DNA duplexes from their original conformations under high pressure depends on the water structure itself and involves displacing an active (structural) water molecule outside the nucleic acid molecules. Experiments carried out until now in many laboratories have shown that B-Z or A-Z transitions of DNA or RNA, respectively, do not depend on the conditions applied, but the common mechanism for these processes seems to be dehydration. This same effect can be observed either at high salt concentrations or in the presence of an alcohol or at high pressure. Our results also support the view that the higher stability of RNA compared with DNA duplexes is due to the strong interaction of the 2'-hydroxyl groups of RNA with water molecules.

Circular Dichroism↗
PubMed1999

Synthesis of infectious viroids and other circular RNAs.

Viroids are small autonomously replicating RNAs that share structural features with other subviral circular single-stranded RNAs of plants. Viroids and other circular single-stranded RNAs can be synthesised in vitro by a PCR-based procedure using a simple set of reactions. Two end-to-end primers are selected from a desired region of the viroid, one for the synthesis of the first strand cDNA and another for the production of the second strand DNA. The second primer contains an 18 nucleotide T7 promoter at its 5' end, and is selected such that the G nucleotide at the transcription start site represents a G in the viroid. Linked reverse transcription-PCR results in linear double-stranded DNA consisting of the viroid sequence and the T7 promoter. Run-off transcription of the PCR product allows the synthesis of exact-length linear viroid RNA which can be circularised by T4 RNA ligase following an enzymic modification of the 5' triphosphate to a monophosphate. This procedure results in authentic viroid molecules and obviates the need for construction and cloning of DNA in the form of tandem repeats for infectivity tests. It also allows PCR-based manipulation of circular RNAs, thus greatly simplifying structure-function analyses of viroid molecules.

DNA, Complementary↗
PubMed1978

alpha-Helix-double helix interaction shown in the structure of a protamine-transfer RNA complex and a nucleoprotamine model.

Single crystal X-ray diffraction and circular dichroism studies of protamine binding to a tRNA suggest that the protamine molecule changes its conformation from a random coil to a structure containing a helices on binding to tRNA, and that alpha-helical segment(s) of protamine bind approximately along a shallow groove of a double-helical portion of ttrna. based on these observations, a structural model for nucleoprotamine is proposed.

Amino Acid Sequence↗
PubMed1976

Mapping of poly(A) sequences in the electron microscope reveals unusual structure of type C oncornavirus RNA molecules.

We have synthesized a convenient electron microscope label for mapping poly(A) sequences. Short lengths of poly(dT) are polymerized onto nicked circular SV40 DNA with the enzyme terminal deoxynucleotidyl transferase. An RNA or DNA molecule of interest is treated with glyoxal, hybridized briefly with the poly(dT) circles, and spread for microscopy; poly(A) stretches are clearly marked because they are attached to the poly(dT) on the easily recognized SV40 duplex circles. The RNAs of several type C oncornaviruses were examined by this method. The endogenous feline virus(RD-114), the endogenous baboon virus (BKD), and the woolly monkey sarcoma virus (WoMV) all contain a dimer of RNA subunits held together in a central secondary structure feature we call the dimer linkage structure. Both ends distal to the dimer linkage structure hybridize to the SV40-poly(dT). Assuming both poly(A)s are on the 3' ends of the subunits and that both subunits are identical, the two identical subunits are held together by interactions between sequences close to the 5' ends.

Avian Sarcoma Viruses↗
PubMed1989

Molecular cloning and terminal sequence determination of the S and M RNAs of tomato spotted wilt virus.

Complementary DNA to the genomic RNA of tomato spotted wilt virus (TSWV) was synthesized and cloned in either pUC19 or lambda gt10. Restriction endonuclease maps were constructed from cDNA clones specific for the S and the M RNA segments, extending 3.0 and 5.4 kbp respectively. The nucleotide sequences of the 3' and 5' termini of both RNA molecules were determined. The S and M RNAs contain inverted complementary repeats at their termini, probably involved in RNA replication and in the formation of circular nucleocapsids in virions. The terminal structures of the TSWV genome resemble, in these aspects, those of the Bunyaviridae.

Base Sequence↗
PubMed1999

Properties of circular dumbbell RNA/DNA chimeric oligonucleotides containing antisense phosphodiester oligonucleotides.

We have designed a new class of oligonucleotides, "dumbbell RNA/DNA chimeric phosphodiesters", containing two alkyl loop structures with RNA/DNA base pairs (sense (RNA) and antisense (DNA) in the double helical stem. The reaction of nicked (NDRDON) and circular (CDRDON) dumbbell RNA/DNA chimeric oligonucleotides with RNaseH gave the corresponding antisense phosphodiester oligonucleotide together with the sense RNA cleavage products. The liberated antisense phosphodiester oligodeoxynucleotide was bound to the target 35mer RNA, which gave 35mer RNA cleavage products by treatment with RNaseH. The circular dumbbell RNA/DNA chimeric oligonucleotide showed more nuclease resistance than the linear antisense phosphodiester oligodeoxynucleotide(anti-ODN) and the nicked dumbbell RNA/DNA chimeric oligonucleotide.

Base Pairing↗
PubMed1982

Acquisition of native conformation of ribosomal 5S ribonucleic acid from Escherichia coli. Hydrodynamic and spectroscopic studies on the unfolding and refolding of ribonucleic acid.

In a continuing effort to decipher the molecular mechanism of ribosome self-assembly [e.g., Dunn, J. M., & Wong, K.-P. (1979) Biochemistry 18, 4380-4385], the mechanism of folding of 5S RNA was investigated by unfolding and refolding studies using several physical techniques including circular dichroism (CD), UV absorption spectroscopy, and sedimentation velocity analysis to monitor various conformational changes. The 5S RNA was unfolded by using 6 M urea and EDTA, and an unfolded state was characterized in which the base pairing was found to be disrupted, but extensive base stacking remained. The unfolded 5S RNA was then refolded upon removal of urea and EDTA by dialysis against a reconstitution buffer both with and without Mg2+, and the refolded states were characterized. The results indicate that under the proper conditions, 5S RNA refolds to a conformation and overall shape very similar to the native conformation. These results indicate that the nucleotide sequence in 5S RNA contains the necessary information to direct the folding of the RNA into its native conformation. The presence of an appropriate concentration of Mg2+ and an incubation at 60 degrees C are required for the correct refolding, since omission of either one results in a renatured 5S RNA whose conformation is quite different from the native one.

Circular Dichroism↗
PubMed2004

Circular viral DNA detection and junction sequence analysis from PBMC of SHIV-infected cynomolgus monkeys with undetectable virus plasma RNA.

Extrachromosomal forms of human immunodeficiency virus (HIV)-1 can be detected in peripheral blood mononuclear cell (PBMC) from HIV-infected patients in the absence of detectable viral replication and are thought to be a sign of active but cryptic virus replication. No information, however, are available on whether these forms are also present in animal models for acquired immunodeficiency syndrome (AIDS) and on their relation with other methods of detection of virus replication. To this aim, a polymerase chain reaction (PCR) approach was used to detect and analyze unintegrated circular 2-LTR-containing forms in PBMC of simian human immunodeficiency virus (SHIV)89.6P infected cynomolgus monkeys with RNA levels ranging between 1.8 x 10(6) and less than 50 copies/ml of plasma. 2-LTR forms were detected in 96.5% of monkeys' samples above 50 copies/ml of plasma, whereas they were present in 75.8% of monkeys' samples below 50 copies/ml of plasma. Persistence of unintegrated viral DNA in monkeys with undetectable plasma RNA could indicate either stability in non-dividing cells or ongoing low levels of viral replication in dividing cells.

Animals↗
PubMed1978

Changes in the conformation and stability of 5 S RNA upon the binding of ribosomal proteins.

The binding of ribosomal proteins L25, L18, and L5 to 5 S RNA results in a conformational change and a destabilization of the 5 S RNA molecule. The changes observed in the near ultraviolet circular dichroism (CD) spectra and in the melting profiles indicate an increase in base stacking uith an accompanying increase in asymmetry of the bases and a decrease in the conformational stability of the 5 S RNA. These results are consistent with the interpretation that the binding of these proteins increases the stacking of specific single-stranded bases in 5 S RNA and aligns them in helical arrays, resulting in a conformation which facilitates base-pairing with nucleotide segment(s) of the ribosomal 23 S RNA or the transfer RNA (or both). The simple and precise difference CD method described here is potentially useful for studying subtle conformational changes of other nucleic acid-protein interactions.

Bacterial Proteins↗
PubMed2004

Exploring Hoogsteen and reversed-Hoogsteen duplex and triplex formation with tricyclo-DNA purine sequences.

The duplex- and triplex-formation properties of the tricyclo-DNA purine decamer 5'p-gagaaggaaa-3' as a single strand or as part of a hairpin duplex with corresponding parallel and antiparallel pyrimidine DNA and RNA complements, as well as with antiparallel purine DNA and RNA complements, were investigated by UV melting curve analysis, circular dichroism spectroscopy, and gel mobility shift experiments. It was found that tricyclo-DNA forms very stable duplexes with the pyrimidine RNA and DNA complements not only in the Watson-Crick pairing mode, but also in the Hoogsteen one. Below pH 6.0, the tc-DNA/DNA and tc-DNA/RNA Hoogsteen duplexes were found to be more stable than the corresponding Watson-Crick DNA duplexes. Triplexes of the hairpin structure with parallel pyrimidine complements revealed even stronger Hoogsteen pairing relative to the duplexes, presumably due to structural preorganization phenomena. Triplex formation with antiparallel pyrimidine and purine third strands (reversed-Hoogsteen motif) could not be observed and seem to be unstable.

Base Sequence↗
PubMed2004

Transcription arrest caused by long nascent RNA chains.

The transcription process is highly processive. However, specific sequence elements encoded in the nascent RNA may signal transcription pausing and/or termination. We find that under certain conditions nascent RNA chains can have a strong and apparently sequence-independent inhibitory effect on transcription. Using phage T3 RNA polymerase (T3 RNAP) and covalently closed circular (cccDNA) DNA templates that did not contain any strong termination signal, transcription was severely inhibited after a short period of time. Less than approximately 10% residual transcriptional activity remained after 10 min of incubation. The addition of RNase A almost fully restored transcription in a dose dependent manner. Throughout RNase A rescue, an elongation rate of approximately 170 nt/s was maintained and this velocity was independent of RNA transcript length, at least up to 6 kb. Instead, RNase A rescue increased the number of active elongation complexes. Thus transcription behaved as an all-or-none process. The mechanism of transcription inhibition was explored using electron microscopy and further biochemical experiments. The data suggest that multiple mechanisms may contribute to the observed effects. Part of the inhibition can be ascribed to the formation of R-loops between the nascent RNA and the DNA template, which provides "roadblocks" to trailing T3 RNAPs. Based on available literature we discuss possible in vivo implications of the results.

DNA↗
PubMed1984

RNA-dependent DNA polymerase activity in cauliflower mosaic virus-infected plant leaves.

Cauliflower mosaic virus (CaMV) is a plant DNA with an 8-kb circular double-stranded genome. CaMV-specific DNA and RNA molecules present in infected Brassica cells share some structural features with DNAs and RNAs of retroviruses and hepatitis B virus. This led to the hypothesis that CaMV replication occurs via reverse transcription of an RNA intermediate. Here we report the first characterization of a new DNA polymerase activity, specific to CaMV-infected tissues. A subcellular fraction of infected cells shows capacity to copy poly(C) and the heteropolymeric regions of natural mRNAs. Chromatographic isolation of the poly(C)-dependent activity clearly establishes that it is distinct from the classical gamma-like DNA polymerases previously described in plant cells. The significant homology observed between defined regions of the Moloney murine leukemia virus (MMLV) polymerase and CaMV unassigned gene V product favours the idea that the reverse transcriptase-like DNA polymerase detected in infected cells is a virus-encoded enzyme.

Journal Article↗
PubMed2004

Role of the trans-activation response element in dimerization of HIV-1 RNA.

The HIV-1 genome consists of two identical RNA strands that are linked together through non-covalent interactions. A major determinant for efficient dimerization of the two RNA strands is the interaction between palindromic sequences in the dimerization initiation site. Here we use an interplay of bioinformatics, biochemistry, and atomic force microscopy to describe another conserved palindrome in the trans-activation response element (TAR) that functions as a strong dimerization site when transiently exposed to the viral nucleocapsid protein. In conjunction with the DIS interaction, the TAR dimerization induces the formation of a 65-nm higher-order circular structure in the dimeric HIV-1 RNA. Our results provide a molecular model for the role of TAR in packaging and reverse transcription of the viral genome. The unique structure of the TAR-TAR dimer renders it an intriguing therapeutic target for the treatment of HIV-1 infection.

5' Untranslated Regions↗