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PubMed1990

Template supercoiling by a chimera of yeast GAL4 protein and phage T7 RNA polymerase.

Fusion of the DNA-binding domain of yeast GAL4 protein to the amino terminus of bacteriophage T7 RNA polymerase yields a chimera that retains the characteristics of its components. The presence of the GAL4 peptide allows the chimeric enzyme to anchor itself on the DNA template, and this anchoring in turn drives the formation of a supercoiled DNA loop, in linear or circular templates, when RNA synthesis at the polymerase site forces a translocation of the DNA relative to the site. Nonspecific interaction between the chimeric enzyme and DNA appears to be sufficient to effect supercoiling during transcription. Transcription by the chimeric polymerase is strictly dependent on the presence of a T7 promoter; thus it provides a tool in vitro and in vivo for specifically supercoiling DNA segments containing T7 promoter sequences.

Amino Acid Sequence↗

PubMed1996

Single stand targeted triplex formation: physicochemical and biochemical properties of foldback triplexes.

Oligodeoxyribonucleotides containing both Watson-Crick and Hoogsteen hydrogen bonding domains joined by a nucleotide loop (FTFOs) are studied for their binding affinity and specificity to the DNA and RNA single-stranded targets. Thermal denaturation studies reveal that FTFOs have high binding affinity for their targets than do antisense (duplex forming) and antigene (triplex forming) oligonucleotides, because of involvement of both the Watson-Crick and Hoogsteen domains in the interaction. Studies with FTFOs containing different sizes and sequences of loops show that 4-6 bases long loops are optimum for binding; loop sequence does not have a dramatic effect on binding. The FTFOs have greater sequence specificity than do antisense and antigene oligonucleotides because they read the target sequence twice. SI-, PI- and mung bean nuclease protection assays show that the DNA FTFO forms a stable triplex with the DNA target strand, but a weak or no triplex with the RNA target strand. Gel mobility shift assay is used to determine binding of FTFOs to DNA and RNA targets. The circular dichroism (CD) spectrum of the foldback triplex formed with the DNA target strand resembles the B-DNA spectrum, suggesting that the triplex has a B-type of conformation.

Binding Sites↗

PubMed1999

Mimicry of the hepatitis delta virus replication cycle mediated by synthetic circular oligodeoxynucleotides.

BACKGROUND: Hepatitis delta virus (HDV) is a circular single-stranded RNA pathogen whose monomeric form results from self-processing. Although studies have examined minimal HDV ribozyme activities, the mechanism for forming the circular virus remains unclear, and the trans catalytic properties of self-processed forms of HDV ribozymes have not been studied. In addition, HDV ribozymes have not previously been engineered to cleave a non-HDV sequence. RESULTS: Long repeating RNAs have been produced from in vitro rolling-circle transcription of synthetic circular oligodeoxynucleotides encoding catalytically active subsets of the entire antigenomic RNA virus. Like full-length HDV, these multimeric RNAs undergo self-processing to monomer length; importantly, cyclization is found to occur efficiently, but only in the presence of the circular template. Linear and circular monomer ribozymes and engineered variants are shown to be active in cleaving HDV and HIV RNA targets in trans, despite having self-binding domains. CONCLUSIONS: Mimicry of the rolling-circle replication pathway for HDV replication has led to a new proposal for cyclization of HDV RNA. Under these conditions, cyclization is mediated by the complementary circular template. In addition, it has been shown that self-processed HDV ribozymes can be catalytically active in trans despite the presence of antisense sequences built into their structure.

Base Sequence↗

PubMed1999

Thermodynamics of RNA hairpins containing single internal mismatches.

Thermodynamic parameters and circular dichroism spectra are presented for RNA hairpins containing single internal mismatches in the stem regions. Three different sequence contexts for the G*U mismatch and two contexts for C*A, G*A, U*U, A*C and U*G mismatches were examined and compared with Watson-Crick base-pair stabilities. The RNA hairpins employed were a microhelix and tetraloop representing the Escherichia coli tRNAAlaacceptor stem and sequence variants that have been altered at the naturally occurring G*U mismatch site. UV melting studies were carried out under different conditions to evaluate the effects of sodium ion concentration and pH on the stability of mismatch-containing hairpins. Our main findings are that single internal mismatches exhibit a range of effects on hairpin stability. In these studies, the size and sequence of the loop and stem are shown to influence the overall stability of the RNA, and have a minor effect on the relative mismatch stabilities. The relationship of these results to RNA-ligand interactions involving mismatch base-pairs is discussed.

Circular Dichroism↗

PubMed1996

Specific interaction of glyceraldehyde 3-phosphate dehydrogenase with the 5'-nontranslated RNA of hepatitis A virus.

Initiation of translation of hepatitis A virus (HAV) RNA occurs by internal entry and is likely to involve the interaction of trans-acting cellular protein factors with cis-acting structural elements of an internal ribosomal entry segment (IRES) within the 5'-nontranslated RNA. To characterize interactions between African green monkey kidney (BS-C-1) cell proteins and the predicted stem-loop IIIa (nucleotides 155-235) located at the 5' border of the HAV IRES, we utilized an electrophoresis mobility shift assay (EMSA) to identify a 39-kDa RNA-binding protein (p39). Amino-terminal amino acid sequencing of highly purified p39 revealed absolute identity with human glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The identity of p39 as simian GAPDH was further confirmed by antigenic and biochemical similarities between p39 and human GAPDH. Analysis of the RNA binding properties of simian GAPDH revealed that this cellular protein interacts with two additional sites in the HAV 5'-nontranslated RNA, one located between nucleotides 1-148 and the other between nucleotides 597-746. Competitive EMSAs also demonstrated that GAPDH and human polypyrimidine tract-binding protein, a putative picornavirus translation initiation factor, compete with each other for binding to stem-loop IIIa, suggesting that the relative cytoplasmic abundance of GAPDH and polypyrimidine tract-binding protein in individual cell-types may be an important determinant of viral translation activity. Human GAPDH was found to destabilize the folded structure of the stem-loop IIIa RNA based upon observed decreases in the circular dichroism spectra of this RNA following binding of the protein. This RNA helix-destabilizing activity of GAPDH could directly influence IRES-dependent translation and/or replication of picornavirus RNA.

Amino Acid Sequence↗

PubMed1990

Isolation of a subterranean clover mottle virus-like satellite RNA from lucerne infected with lucerne transient streak virus.

A circular, viroid-like satellite RNA (sat RNA) was detected in lucerne transient streak virus (LTSV), from lucerne in South Australia. It was larger than the previously reported sat RNA of LTSV, being similar in size and sequence homology to the 388 nucleotide sat RNA previously shown to be encapsidated by subterranean clover mottle virus (SCMoV) isolated from subterranean clover in Western Australia. This indicates that under field conditions, very similar sat RNAs can be associated with two distantly related sobemoviruses, LTSV and SCMoV. The natural hosts of these viruses are lucerne and subterranean clover, respectively.

Australia↗

PubMed1995

Intra- and intermolecular nonenzymatic ligations occur within transcripts derived from the peach latent mosaic viroid.

We report here the nonenzymatic self-ligation of transcripts corresponding to the peach latent mosaic viroid (PLMVd). This is the first description of this process with viroid sequences, although it has been reported to occur with human hepatitis delta virus RNA. Self-ligation occurs when the 5'-hydroxyl and the 2',3'-cyclic phosphate termini produced by the hammerhead self-cleavage of the viroid RNA are juxtaposed by the viroid rod-like structure, and a phosphodiester bond is formed between the two following hydrolysis of the cyclic phosphate. Unit-length transcripts undergo intramolecular folding, and their subsequent self-ligation produces circular molecules. The self-ligation observed in vitro may contribute to PLMVd circularization during rolling circle replication; however, this does not exclude the possibility that a host RNA ligase catalyzes the ligation steps in vivo. Like self-cleavage, self-ligation is probably an ancestral reaction, and the enzyme-catalyzed ligation most likely evolved from this primitive mechanism. Furthermore, the intermolecular self-ligation of annealed transcripts derived from PLMVd is demonstrated, suggesting a possible mechanism for sequence reassortment in viroids.

Base Sequence↗

PubMed1986

A self-splicing RNA excises an intron lariat.

We have investigated the in vitro self-splicing of a class II mitochondrial intron. A model pre-mRNA containing intron 5 gamma of the oxi 3 gene of yeast mitochondrial DNA undergoes an efficient intramolecular rearrangement reaction in vitro. This reaction proceeds under conditions distinct from those optimal for self-splicing of class I introns, such as the Tetrahymena nuclear rRNA intron. Intron 5 gamma is excised as a nonlinear RNA indistinguishable from the in vivo excised intron product by gel electrophoresis and primer extension analysis. Studies of the in vitro excised intron product strongly indicate that it is a branched RNA with a circular component joined by a linkage other than a 3'-5' phosphodiester. Two other products, the spliced exons and the broken form of the lariat, were also characterized. These results show that the class II intron products are similar to those of nuclear pre-mRNA splicing.

DNA, Mitochondrial↗

PubMed1986

Structure and replication of the genome of the hepatitis delta virus.

The hepatitis delta virus can be found in the serum and liver of some hepatitis B virus patients. We now report that the RNA genome of serum-derived delta virus is single-stranded and circular. Livers of infected chimpanzees or woodchucks contained as many as 300,000 copies of genomic strand RNA per average cell, and at least some of this RNA had a circular conformation. Also present in the livers were RNA species complementary to the virion RNA. The genomic RNA was 5-22 times more abundant than this antigenomic strand. Some of the antigenomic RNA was complexed with genomic RNA, as evidenced by the fact that at least 34% of the antigenomic RNA was resistant to digestion with either RNase A in 0.3 M NaCl or S1 nuclease. Some of the antigenomic RNA was in a circular conformation. These and other findings showed that the structure and replication of hepatitis delta virus are in many ways similar to those of the previously described plant viroids, virusoids, and satellite RNAs.

Animals↗

PubMed1991

High-resolution NMR studies of chimeric DNA-RNA-DNA duplexes, heteronomous base pairing, and continuous base stacking at junctions.

Two symmetrical DNA-RNA-DNA duplex chimeras, d(CGCG)r(AAUU)d(CGCG) (designated rAAUU) and d(CGCG)r(UAUA)d(CGCG) (designated rUAUA), and a nonsymmetrical chimeric duplex, d(CGTT)r(AUAA)d(TGCG)/d(CGCA)r(UUAU)d(A ACG) (designated rAUAA), as well as their pure DNA analogues, containing dU instead of T, have been synthesized by solid-phase phosphoramidite methods and studied by high-resolution NMR techniques. The 1D imino proton NOE spectra of these d-r-d chimeras indicate normal Watson-Crick hydrogen bonding and base stacking at the junction region. Preliminary qualitative NOESY, COSY, and chemical shift data suggest that the internal RNA segment contains C3'-endo (A-type) sugar conformations except for the first RNA residues (position 5 and 17) following the 3' end of the DNA block, which, unlike the other six ribonucleotides, exhibit detectable H1'-H2' J coupling. The nucleosides of the two flanking DNA segments appear to adopt a fairly normal C2'-endo B-DNA conformation except at the junction with the RNA blocks (residues 4 and 16), where the last DNA residue appears to adopt an intermediate sugar conformation. The DNA-RNA junction residues exhibit quite different COSY, chemical shift, and NOE behavior, but these effects do not appear to propagate into the DNA or RNA segments. The circular dichroism spectra of these d-r-d chimeras also display a mixture of characteristic A-type and B-type absorption bands. The data indicate that A-type and B-type conformations can coexist in a single short continuous nucleic acid duplex, but our results differ somewhat from previous theoretical model studies.

Base Sequence↗

PubMed1995

Nevirapine alters the cleavage specificity of ribonuclease H of human immunodeficiency virus 1 reverse transcriptase.

The action of the dipyridodiazepinone nevirapine (BI-RG-587) on polymerization and RNase H activities of human immunodeficiency virus reverse transcriptase (RT) was examined. Substrates using heteropolymeric DNA primers hybridized to complementary RNA templates were employed. Challenged assays were performed that allowed measurement of activity of the RT resulting from a single round of binding of RT to substrate. Results demonstrated that nevirapine alters the cleavage specificity of the RNase H. Instead of a primary cleavage approximately 18 nucleotides upstream of the DNA 3' terminus, multiple cleavages were observed ahead of and behind this site. This indicated that the compound facilitates sliding of the RT away from the DNA primer terminus allowing cleavage at more sites. The change in specificity occurred whether the primer terminus was at the end or internal on the template. Experiments with RNA primers on circular DNA demonstrated a nevirapine-induced stimulation of RNase H activity beyond the increase expected from the change in cleavage specificity. Examination of polymerization showed that the compound decreased both the number of primers that underwent synthesis and the processive elongation of those primers. The significance of these results with respect to viral replication and recombination is discussed.

Base Sequence↗

PubMed1998

Triplex-directed self-assembly of an artificial sliding clamp on duplex DNA.

BACKGROUND: Circular triplex-forming oligonucleotides (CTFOs) have previously been shown to bind tightly to short single-stranded homopurine DNAs in a sequence-specific manner. In view of the importance of double-stranded DNA as a target in the development of gene-specific therapeutic and diagnostic agents, we have investigated the binding of CTFOs to double-helical DNA. RESULTS: DNA-binding experiments show that a CTFO can recognize its homopurine target when the target is embedded in a long duplex. Unlike their linear counterparts, CTFOs bind the double helix in two topologically distinct forms. The more stable of the two complexes is found to be a pseudorotaxane, having the same topology as the sliding clamp protein subunits associated with some DNA and RNA polymerases. CONCLUSIONS: Circular triplex-forming oligonucleotides have been shown to bind the DNA double helix in a topological manner which is unprecedented among synthetic ligands. This novel binding motif allows a synthetic CTFO to be irreversibly locked onto a circular double-stranded DNA target without covalently modifying the target.

Binding Sites↗

PubMed1992

Circular dichroism and molecular modeling yield a structure for the complex of human immunodeficiency virus type 1 trans-activation response RNA and the binding region of Tat, the trans-acting transcriptional activator.

Transcription in the human immunodeficiency virus type 1 (HIV-1) retrovirus is regulated by binding the viral Tat protein (trans-acting transcriptional activator) to the trans-activation response (TAR) RNA sequence. Here, vacuum UV circular dichroism (VUV-CD) is used to study the structure of TAR and its complex with two peptide fragments that are important for Tat binding to TAR. The VUV-CD spectrum of TAR is typical of A-form RNA and is minimally perturbed when bound to either the short or the long Tat peptide. The CD spectra of the complexes indicate an extended structure in the arginine-rich region of Tat from amino acid residue 47 through residue 58 and a short alpha-helix within the adjacent 59-72 region. Models of TAR and its peptide complexes are constructed to integrate these spectroscopic results with current biochemical data. The model suggests that (i) the arginine-rich 49-58 region is primarily responsible for electrostatic interactions with the phosphates of the RNA, (ii) the arginine side chains can additionally interact with substituent groups of the nucleotide bases to confer base recognition in the complex, (iii) the recognition of uracil-23 in TAR is facilitated by the peptide backbone, and (iv) the glutamine-rich face of an alpha-helix within the 59-72 region pairs to bases UGG at nucleotide positions 31-33 in the TAR loop and thus provides an additional motif in the Tat trans-activating protein to recognize TAR RNA.

Amino Acid Sequence↗

PubMed1990

Reverse self-splicing of group II intron RNAs in vitro.

Group II introns, which are classed together on the basis of a conserved secondary structure, are found in organellar genes of lower eukaryotes and plants. Like introns in nuclear pre-messenger RNA, they are excised by a two-step splicing reaction to generate branched circular RNAs, the so-called lariats. A remarkable feature of group II introns is their self-splicing activity in vitro. In the absence of a nucleotide cofactor, the intron RNAs catalyse two successive transesterification reactions which lead to autocatalytic excision of the lariat IVS from pre-mRNA and concomitantly to exon ligation. By virtue of its ability to specifically bind the 5' exon, the intron can also catalyse such reactions on exogenous RNA substrates. This sequence-specific attachment could enable group II introns to integrate into unrelated RNAs by reverse splicing, in a process similar to that described for the self-splicing Tetrahymena group I intron. Here we report that group II lariat IVS can indeed reintegrate itself into an RNA composed of the ligated exons in vitro. This occurs by a process of self-splicing that completely reverses both transesterification steps of the forward reaction: it involves a transition of the 2'-5' phosphodiester bond of the lariat RNA into the 3'-5' bond of the reconstituted 5' splice junction.

Apoproteins↗

PubMed1991

Conformational studies on a peptide fragment representing the RNA-binding N-terminus of a viral coat protein using circular dichroism and NMR spectroscopy.

Conformational studies were performed on a synthetic pentacosapeptide representing the RNA-binding N-terminal region of the coat protein of cowpea chlorotic mottle virus. The effects of ionic strength, addition of (oligo)phosphates and temperature on the conformation of this highly positively charged peptide containing six arginines and three lysines were studied. CD experiments show that the peptide has 15-18% alpha-helical conformation and about 80% random-coil conformation in the absence of inorganic salt at 25 degrees C, and 20-21% alpha-helical conformation under the same conditions at 10 degrees C. Addition of inorganic salts results in an increase of alpha-helix content, up to 42% in the presence of oligophosphate with an average chain length of 18 phosphates, which was used as an RNA analog. NMR experiments show that the alpha-helix formation starts in the region between Thr9 and Gln12, and is extended in the direction of the C terminus. Relaxation measurements show that binding to oligophosphates of increasing length results in reduced internal mobilities of the positively charged side chains of the arginyl and lysyl residues and of the side chain of Thr9 in the alpha-helical region. The alpha-helix formation in the N-terminal part of this viral coat protein upon binding of phosphate groups to the positively charged side chains is suggested to play an essential role in RNA binding.

Amino Acid Sequence↗

PubMed1998

Inhibition of Cellular RNA polymerase II transcription by delta antigen of hepatitis delta virus.

Hepatitis delta virus (HDV) contains a circular, viroid-like RNA and the hepatitis delta antigen (HDAg) protein. The viral RNA is replicated via RNA-dependent RNA synthesis, which is thought to be mediated by host DNA-dependent RNA polymerase II (pol II). The precise mechanism of HDV RNA replication using RNA as a template remains to be elucidated, though it is clear that HDAg is involved. We demonstrate here that both SP1-activated and basal pol II transcription are inhibited by HDAg. This inhibitory effect of HDAg was observed in vivo in transient cotransfection assays as well as in vitro in HeLa nuclear extracts with purified, recombinant HDAg. The in vitro inhibition of pol II transcription could be reversed with excess HeLa nuclear extracts. Furthermore, HDAg specifically inhibited pol II-mediated transcription but not pol I- or III-mediated transcription. These results provide support for the model in which HDAg participates in a complex with host cell pol II transcription factors to mediate pol II-dependent HDV RNA replication, concomitantly cellular pol II transcription.

Chloramphenicol O-Acetyltransferase↗

PubMed1997

Analysis of the tertiary structure of the ribonuclease P ribozyme-substrate complex by site-specific photoaffinity crosslinking.

Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein containing a catalytic RNA. The secondary structure of this ribozyme is well-established, and a low-resolution model of the three-dimensional structure of the ribozyme-substrate complex has been proposed based on site-specific crosslinking and phylogenetic comparative data [Harris ME et al., 1994 EMBO J 13:3953-3963]. However, several substructures of that model were poorly constrained by the available data. In the present analysis, additional constraints between elements within the Escherichia coli RNase P RNA-pre-tRNA complex were determined by intra- and intermolecular crosslinking experiments. Circularly permuted RNase P RNAs were used to position an azidophenacyl photoactive crosslinking agent specifically at strategic sites within the ribozyme-substrate complex. Crosslink sites were mapped by primer extension and confirmed by analysis of the mobility of the crosslinked RNA lariats on denaturing acrylamide gels relative to circular and linear RNA standards. Crosslinked species generally retained significant catalytic activity, indicating that the results reflect the native ribozyme structure. The crosslinking results support the general configuration of the structure model and predicate new positions and orientations for helices that were previously poorly constrained by the data set. The expanded library of crosslinking constraints was used, together with secondary and tertiary structure identified by phylogenetic sequence comparisons, to refine significantly the model of RNase P RNA with bound substrate pre-tRNA. The crosslinking results and data from chemical-modification and mutational studies are discussed in the context of the current structural perspective on this ribozyme.

Affinity Labels↗

PubMed1993

DNA packaging ATPase of bacteriophage T3.

A defined in vitro DNA packaging system of phage T3, which is composed of purified proheads and two packaging proteins, the products of genes 18 and 19 (gp18 and gp19, respectively), displayed a DNA-dependent ATPase activity. ATP was hydrolyzed to ADP and Pi. The ATPase activity was stimulated by nonpackageable DNA, such as single-stranded or circular DNA, or RNA (nonpac-ATPase). Among the inhibitors of DNA packaging, actinomycin D specifically inhibited the ATPase activity that was tightly coupled to DNA packaging (pac-ATPase), but did not inhibit the nonpac-ATPase activity. Both activities depended upon a functional packaging complex, but the nonpac-ATPase, once activated, did not require DNA. Unpackageable pUC18 DNA inhibited the pac-ATPase and the phage yield in parallel. Approximately one molecule of ATP was hydrolyzed during the translocation of 1.8 bp of T3 DNA.

Adenosine Triphosphatases↗