Replication of RNA viruses: analysis by liquid polymer phase partition of the binding of Q RNA polymerase to nucleic acid.
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Binding Sites↗
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Restriction endonuclease mapping of the DNA of Rous-associated virus O reveals extensive homology in structure and sequence with avian sarcoma virus DNA.
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Alpharetrovirus↗
RNA polymerase assembly in vitro. Temperature dependence of reactivation of denatured core enzyme.
The Escherichia coli RNA polymerase core molecule, after denaturation in 6 M guanidine hydrochloride, can be completely reactivated in the absence of sigma subunit. Reactivation is temperature dependent. At 4 degrees a renatured-inactive preparation is formed that has most of the secondary structure of the original native molecule but has a reduced sedimentation coefficient and a smaller Stokes radius and is, therefore, of lower molecular weight. Upon warming to 37 degrees the renatured-inactive preparation is converted in a time-dependent process to the renatured-active preparation, which has the same amount of secondary structure and same molecular weight as native RNA polymerase. Since the renatured-inactive material is probably composed of subunit assemblies and can be readily reactivated, it should be useful for studying the subunit interactions and control of assembly of RNA polymerase.
Binding Sites↗
Molecular characterisation of the enolase gene from the human malaria parasite Plasmodium falciparum. Evidence for ancestry within a photosynthetic lineage.
We have isolated and characterised the gene encoding the glycolytic enzyme enolase (2-phospho-D-glycerate hydrolase) from the human malaria parasite Plasmodium falciparum. This was achieved using a combination of cDNA sequencing and inverse-PCR techniques. The gene maps to chromosome 10 of the parasite. We have also mapped two further glycolytic enzyme genes, glyceraldehyde-3-phosphate dehydrogenase and triose-phosphate isomerase, to chromosome 14. The enolase gene encodes a protein of 446 amino acids (48.7 kDa), and all amino acid residues implicated in substrate/cofactor binding and catalysis are conserved in the malarial enolase molecule. The predicted protein sequence displays approximately 60-70% identity to enolase molecules of other eukaryotes, the closest relationship with its homologues seen amongst the seven fully described glycolytic pathway enzymes of P. falciparum. Of particular significance in this well conserved molecule is a characteristic 5-amino-acid insertion sequence that is identical in position and virtually identical in primary structure to that which is otherwise found uniquely in plant enolase proteins. This pentapeptide, together with other features of the plasmodial sequence, points to a common ancestry with photosynthetic organisms at the level of a protein-encoding nuclear gene, thus extending earlier analyses of nuclear small-subunit ribosomal RNA genes, and of an extrachromosomal circular 35-kb DNA element found in P. falciparum, which have also indicated such a relationship.
Amino Acid Sequence↗
Involvement of ribosomal ribonucleic acid operons in Salmonella typhimurium chromosomal rearrangements.
As part of our efforts to understand factors influencing chromosomal organization and rearrangements, we studied a family of Salmonella typhimurium tandum duplication mutants. We found that the duplications were originally generated by unequal recombination between pairs of similarly oriented ribosomal ribonucleic acid operons (rrn). This demonstration involved the physical isolation of the duplicated material as circular deoxyribonucleic acid excised from the duplication. The four rrn operons involved embraced the ilv pur D segment of the chromosome and occurred at positions closely analogous to those previously observed for Escherichia coli. The interval between rrnC and rrnA of S. typhimurium was similar in size to that of E. coli (43 versus 39 kilobases), as was the interval between rrnB and rrnE (94 versus 91 kilobases). The rrnA-to-rrnB interval of S. typhimurium, however, was 155 kilobases, substantially greater than the 126 kilobases observed for E. coli.
Chromosomes, Bacterial↗
A novel cis-acting element facilitates minus-strand DNA synthesis during reverse transcription of the hepatitis B virus genome.
Hepadnaviruses replicate through reverse transcription of an RNA pregenome, resulting in a relaxed circular DNA genome. The first 3 or 4 nucleotides (nt) of minus-strand DNA are synthesized by the use of a bulge in a stem-loop structure near the 5' end of the pregenome as a template. This primer is then transferred to a complementary UUCA motif, termed an acceptor, within DR1* near the 3' end of the viral pregenome via 4-nt homology, and it resumes minus-strand DNA synthesis: this process is termed minus-strand transfer or primer translocation. Aside from the sequence identity of the donor and acceptor, little is known about the sequence elements contributing to minus-strand transfer. Here we report a novel cis-acting element, termed the beta5 region (28 nt in length), located 20 nt upstream of DR1*, that facilitates minus-strand DNA synthesis. The deletion or inversion of the sequence including the beta5 region diminished minus-strand DNA synthesis initiated at DR1*. Furthermore, the insertion of the beta5 region into its own position in a mutant in which the sequences including the beta5 region were replaced restored minus-strand DNA synthesis at DR1*. We speculate that the beta5 region facilitates minus-strand transfer, possibly by bringing the acceptor site in proximity to the donor site via base pairing or by interacting with protein factors involved in this process.
Cell Line, Tumor↗
Hepatitis B virus genotypes: a South Pacific perspective.
87-91% but still, 0.6% of those that did respond to vaccination became infected. The infection rate of the vaccinated populations in the Pacific Islands ranged between 0.7 and 3.8%, which is comparable to Taiwan. A vigorous polyclonal response This communication discusses the current status of research in the hepatitis B virus in relation to the South Pacific. The hepatitis B virus (HBV) is a small DNA virus--3200 nucleotides. It has a circular genome and replicates through an RNA intermediate giving this DNA virus many characteristics similar to RNA viruses. Viral genomes can be single-stranded (+ or - sense) or double-stranded. If not vaccinated, infants born to HBeAg positive mothers (i.e. with high viral titer) have a 90% chance of being infected and becoming HBV carriers themselves. Mutants that affect the major antigenic determinant in HBV surface antigens are probably responsible for HBV infection despite immunization and mutants in the polymerase protein may render HBV resistant to therapy with nucleoside analogs. Within HBV seven genotypes A-G have been reported that is, HBV genotype A (HBVA), HBV genotype B (HBVB) etc. HBV is endemic worldwide with an estimated that 5% of the worlds population being carriers. Before the introduction of vaccination programs carrier rates varied between 5-30% in communities of these ethnic groups, and in some cases 80-90% of a community tested positive for HBV markers (i.e. were infected or had been infected). In Taiwan, of vaccinated babies born to HBV positive mothers, the proportion of those that responded to vaccination varied between will usually result in an acute infection and viral clearance. An associated problem with HBV, in the South Pacific, is the hepatitis delta virus (HDV). HDV is a satellite viroid-like RNA virus that requires HBV for replication. It can either co-infect with, or super-infect upon HBV infection resulting in acute infection and/or chronic infection respectively.
Carrier State↗
In vitro evidence that transcription-induced stress causes nucleosome dissolution and regeneration.
During transcription, positive and negative superhelical stresses are generated on a DNA template which could potentially affect nucleosomal structure. When transcription was performed on a closed circular plasmid containing nucleosomes, using T7 RNA polymerase and topoisomerase I, nucleosomal structure was lost from the DNA. Nucleosome content was assayed by analyzing both the topological state of the DNA and the nuclease-resistant fragments produced by micrococcal nuclease and DNase I treatment. This nucleosome dissolution required positive superhelical stress as evidenced by the requirement that the extended RNA transcript remain associated with the polymerase during the transcription process. Rates of transcription were found to be independent of whether the nucleosomes dissolved. When transcription was performed in the absence of topoisomerase I, nucleosome reformation occurred very rapidly. This observation suggests that negative superhelical stress, induced in the wake of polymerase action, facilitates nucleosome reformation.
Animals↗
Mutational analysis of bovine viral diarrhea virus RNA-dependent RNA polymerase.
Recombinant bovine viral diarrhea virus (BVDV) nonstructural protein 5B (NS5B) produced in insect cells has been shown to possess an RNA-dependent RNA polymerase (RdRp) activity. Our initial attempt to produce the full-length BVDV NS5B with a C-terminal hexahistidine tag in Escherichia coli failed due to the expression of insoluble products. Prompted by a recent report that removal of the C-terminal hydrophobic domain significantly improved the solubility of hepatitis C virus (HCV) NS5B, we constructed a similar deletion of 24 amino acids at the C terminus of BVDV NS5B. The resulting fusion protein, NS5BDeltaCT24-His, was purified to homogeneity and demonstrated to direct RNA replication via both primer-dependent (elongative) and primer-independent (de novo) mechanisms. Furthermore, BVDV RdRp was found to utilize a circular single-stranded DNA as a template for RNA synthesis, suggesting that synthesis does not require ends in the template. In addition to the previously described polymerase motifs A, B, C, and D, alignments with other flavivirus sequences revealed two additional motifs, one N-terminal to motif A and one C-terminal to motif D. Extensive alanine substitutions showed that while most mutations had similar effects on both elongative and de novo RNA syntheses, some had selective effects. Finally, deletions of up to 90 amino acids from the N terminus did not significantly affect RdRp activities, whereas deletions of more than 24 amino acids at the C terminus resulted in either insoluble products or soluble proteins (DeltaCT179 and DeltaCT218) that lacked RdRp activities.
Amino Acid Sequence↗
Genome organization of retroviruses. V. In vitro-synthesized Moloney murine leukemia viral DNA has long terminal redundancy.
Purified virions of Moloney murine leukemia virus can synthesize genome-length double-stranded DNA in vitro. Two predominant species of long DNA transcripts, with average sizes of 9.1 and 8.5 kilobases (kb) can be identified. Both species of DNA contain the negative (complementary to viral RNA) and positive (same polarity as viral RNA) strands. However, only the negative strand of the 8.5-kb species can be identified if the synthesis of DNA is carried out in the presence of the drug actinomycin D. The 9.1-kb species appears to be slightly larger than the genomic RNA. If the linear double-stranded 9.1-kb species is treated with Escherichia coli exonuclease III and allowed to anneal, circular DNA molecules can be observed. Furthermore, polyadenylate-containing short genomic RNA fragments (0.5 to 1.0 kb) can anneal to both the 5' and the 3' termini of 9.1-kb complementary DNA. The polyadenylate moiety of the RNA fragments can be identified by tagging it with circular polyoma DNA containing polydeoxybromouridylic acid tails. Thus, the 9.1-kb complementary DNA transcript with two circular polyoma DNA molecules at its termini can be observed. However, when similar annealings are performed with 8.5-kb complementary DNA species, only one end of the resulting molecule has circular polyoma DNA. We conclude that the 9.1-kb complementary DNA species has a large terminal redundancy. The sequences involved in terminal redundancy appear to be derived from the 3' end of the genomic RNA.
Cell-Free System↗
The glycine-rich domain of nucleolin has an unusual supersecondary structure responsible for its RNA-helix-destabilizing properties.
Nucleolin, a major nucleolar protein implicated in preribosome assembly and transcriptional regulation, possesses a C-terminal domain unusually rich in glycine, arginine, and phenylalanine residues. A polypeptide (p10), corresponding to this domain, has been synthesized by means of an Escherichia coli expression system and purified to homogeneity. Nitrocellulose binding assays have clearly shown that this domain of nucleolin is capable of interacting with RNA, and indeed all nucleic acids tested, in an efficient but nonspecific manner. A combination of circular dichroism and infrared spectroscopy provide strong evidence that repeated beta-turns are a major structural component of this polypeptide, which is entirely consistent with its amino acid composition and above all the presence of repeat motifs such as RGGF. Circular dichroism technique also shows that the interaction of p10 with RNA involves an unstacking of the nucleotide bases and an unfolding of the RNA secondary structure. While the role of the C-terminal domain of nucleolin in vivo has yet to be established, our findings suggest that it may act to unfold regions of ribosomal RNA so that a second domain of nucleolin has access to its specific binding site.
Amino Acid Sequence↗
Structure of RNA polymerase II promoters. Coordinate conformational alteration of the upstream activator of the TATA- and RNA-initiation sequences under moderate torsional stress.
Different conformations of the circular DNA domains containing the intergenic region of the Saccharomyces cerevisiae GAL1-GAL10 divergent genes (914 base pairs) were modified by in vitro ligation in various conditions. The effect of increasing torsional stress on the conformation of the composing elements was determined by analysis of the sensitivity to the single strand-specific S1 endonuclease and it was observed that the sites of conformational alterations correspond to the positions relevant for promoter function (upstream activator sequence, TATA sequence, and RNA initiation site).
Base Sequence↗
Self-cleavage of virusoid RNA is performed by the proposed 55-nucleotide active site.
Virusoids are circular single-stranded RNAs dependent on plant viruses for replication and encapsidation. Recently, we showed that an in vitro-synthesized RNA containing 273 nucleotides of the 324-nucleotide virusoid of lucerne transient streak virus self-cleaves at a unique site. The reaction requires heating and snap cooling of the RNA and the subsequent addition of magnesium ions. Here, we test the 55-nucleotide, hammerhead-shaped, structural model proposed for the active site by preparing RNAs with both 5' and 3' terminal deletions. Results indicate that the hammerhead structure is sufficient and necessary for self-cleavage, that certain sequences prevent the formation of the hammerhead structure in the native virusoid RNA, and that an RNA molecule containing only 52 nucleotides is capable of an RNA-mediated reaction.
Base Sequence↗
Modified RNAs as potential drug targets.
Bleomycin (BLM) is a natural antibiotic that is effective in treatment of selected cancers. Although the exact therapeutic mechanism of bleomycin is not known, its target is thought to be a nucleic acid. Besides cleaving DNA, in vitro, Fe-bleomycin cleaves the anticodon of yeast tRNA(Phe) specifically. Using CD and fluorescence spectroscopy we have found that apo-bleomycin binds to synthetic RNA analogs of the anticodon of yeast tRNA(Phe) with an affinity similar to that previously reported for DNA. In order to understand BLM's selectivity, the role magnesium ions play in RNA recognition should be explained. Many RNA substrates for Fe-BLM, including yeast tRNA(Phe), are not cleaved by the drug when the Mg2+ concentration exceeds 1 mM. Competition experiments with anticodon analogs provide insight into the role of magnesium ions in RNA recognition by BLM. These simple modified RNAs may be useful as model systems for investigating BLM/RNA recognition and development of highly selective drugs toward RNA targets.
Antibiotics, Antineoplastic↗
Hybridization of 2'-ribose modified mixed-sequence oligonucleotides: thermodynamic and kinetic studies.
In this study, we characterize the thermodynamics of hybridization, binding kinetics and conformations of four ribose-modified (2'-fluoro, 2'-O-propyl, 2'-O-methoxyethyl and 2'-O-aminopropyl) decameric mixed-sequence oligonucleotides. Hybridization to the complementary non-modified DNA or RNA decamer was probed by fluorescence and circular-dichroism spectroscopy and compared to the same duplex formed between two non-modified strands. The thermal melting points of DNA-DNA duplexes were increased by 1.8, 2.2, 0.3 and 1.3 degrees C for each propyl, methoxyethyl, aminopropyl and fluoro modification, respectively. In the case of DNA-RNA duplexes, the melting points were increased by 3.1, 4.1 and 1.0 degrees C for each propyl, methoxyethyl and aminopropyl modification, respectively. The high stability of the duplexes formed with propyl-, methoxyethyl- and fluoro-modified oligonucleotides correlated with high preorganization in these single-strands. Despite higher thermodynamic duplex stability, hybridization kinetics to complementary DNA or RNA was slower for propyl- and methoxyethyl-modified oligonucleotides than for the non-modified control. In contrast, the positively-charged aminopropyl-modified oligonucleotide showed rapid binding to the complementary DNA or RNA.
Base Pairing↗
The trp RNA-binding attenuation protein (TRAP) from Bacillus subtilis binds to unstacked trp leader RNA.
TRAP (trp RNA-binding attenuation protein) is a tryptophan-activated RNA-binding protein that regulates expression of the trp biosynthetic genes by binding to a series of GAG and UAG trinucleotide repeats generally separated by two or three spacer nucleotides. Previously, we showed that TRAP contains 11 identical subunits arranged in a symmetrical ring. Based on this structure, we proposed a model for the TRAP.RNA interaction where the RNA wraps around the protein with each repeat of the RNA contacting one or a combination of two adjacent subunits of the TRAP oligomer. Here, we have shown that RNAs selected in vitro based on their ability to bind tryptophan-activated TRAP contain multiple G/UAG repeats and show a strong bias for pyrimidines as the spacer nucleotides between these repeats. The affinity of the TRAP.RNA interaction displays a nonlinear temperature dependence, increasing between 5 degrees C and 47 degrees C and then decreasing from 47 degrees C to 67 degrees C. Differential scanning calorimetry and circular dichroism spectroscopy demonstrate that TRAP is highly thermostable with few detectable changes in the structure between 25 degrees C and 70 degrees C, suggesting that the temperature dependence of this interaction reflects changes in the RNA. Results from circular dichroism and UV absorbance spectroscopy support this hypothesis, demonstrating that trp leader RNA becomes unstacked upon binding TRAP. We propose that the bias toward pyrimidines in the spacer nucleotides of the in vitro selected RNAs represents the inability of Us and Cs to form stable base stacking interactions, which allows the flexibility needed for the RNA to wrap around the TRAP oligomer.
Bacillus subtilis↗
A study on the transcription of the polyoma viral genome.
The transcription of polyoma (Py) viral RNA was studied in primary mouse kidney cell cultures (a) during normal, asynchronous infection, (b) under conditions where Py-induced DNA synthesis was inhibited with 5-fluorodeoxyuridine, and (c) after the synchronized onset of Py-induced DNA synthesis. The results obtained suggest that the bulk of Py RNA transcribed after the (asynchronous or synchronized) onset of Py-induced DNA synthesis, designated as "late" Py RNA, corresponds to the polycistronic transcript of most or all of the genetic information contained in a strand of circular Py DNA. The rate at which late Py RNA is transcribed is related to the amount of free Py DNA present in the cultures. Py-infected cultures contain a second, minor component of Py RNA (of one or possibly more species), designated as "early" Py RNA. At least some of this RNA continues to be transcribed if Py-induced DNA synthesis is inhibited with 5-fluorodeoxyuridine and, possibly, also late during normal infection.
Centrifugation, Density Gradient↗
Recently replicated simian virus 40 DNA is a preferential template for transcription and replication.
The template activities for the processes of replication and transcription were compared for recently replicated ("new") and uniformly labeled ("old") simian virus 40 (SV40) DNA in infected monkey cells (line TC7). New SV40 DNA (pulse-labeled for 1 h) served as a template for a second round of replication with a relatively high probability (8% of the DNA replicated per h) for a period of 5 h, after which time its template activity rapidly decreased by severalfold. Old SV40 DNA (labeled for 24 h) functioned as a template for replication with a constant probability (1.8% of the DNA replicated per h) for at least 12 h. The proportion of RNA polymerase with nonreplicated and with recently replicated (bromodeoxyuridine-substituted) viral DNA was determined by an assay that used the Triton-soluble SV40 transcription complex. The proportion of RNA polymerase associated with nonreplicated SV40 DNA decreased very slowly (to 50% in 6 h), strongly suggesting that replicating viral genomes are not required as templates for the initiation of late transcription. This hypothesis was supported by the finding that the RNA synthesized in vitro was associated with covalently closed circular SV40 DNA. Furthermore, after 9 h in bromodeoxyuridine, the recently replicated viral DNA had nearly three times more RNA polymerase per unit of DNA than did the nonreplicated DNA. We thus conclude that recently replicated SV40 DNA is utilized preferentially as a template for transcription and for replication.
Cell Line↗