Showing papers on "RNA-dependent RNA polymerase published in 1986"
TL;DR: A gene expression system based on bacteriophage T7 RNA polymerase has been developed and high levels of accumulation suggest that the RNAs are relatively stable, perhaps in part because their great length and/or stem-and-loop structures at their 3' ends help to protect them against exonucleolytic degradation.
6,415 citations
TL;DR: In this paper, the authors describe a method to clone a functional gene for bacteriophage T7 RNA polymerase, which is useful for synthesizing large amounts of RNA in vivo or in vitro, and can produce a single RNA selectively from a complex mixture of DNAs.
Abstract: This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.
971 citations
TL;DR: Evidence is presented for the in vitro autolytic processing of dimeric and trimeric forms of this satellite RNA of tobacco ringspot virus, which apparently is reversible to form dimeric RNA from monomeric RNA, and does not require an enzyme for its catalysis.
Abstract: Associated with some plant viruses are small satellite RNA's that depend on the plant virus to provide protective coat protein and presumably at least some of the proteins necessary for satellite RNA replication. Multimeric forms of the satellite RNA of tobacco ringspot virus are probable in vivo precursors of the monomeric satellite RNA. Evidence is presented for the in vitro autolytic processing of dimeric and trimeric forms of this satellite RNA. The reaction generates biologically active monomeric satellite RNA, apparently is reversible to form dimeric RNA from monomeric RNA, and does not require an enzyme for its catalysis.
552 citations
TL;DR: The shortened form of the self-splicing ribosomal RNA intervening sequence of Tetrahymena thermophila acts as an enzyme in vitro that can act as an RNA polymerase, differing from the protein enzyme in that it uses an internal rather than an external template.
Abstract: A shortened form of the self-splicing ribosomal RNA (rRNA) intervening sequence of Tetrahymena thermophila acts as an enzyme in vitro. The enzyme catalyzes the cleavage and rejoining of oligonucleotide substrates in a sequence-dependent manner with Km = 42 microM and kcat = 2 min-1. The reaction mechanism resembles that of rRNA precursor self-splicing. With pentacytidylic acid as the substrate, successive cleavage and rejoining reactions lead to the synthesis of polycytidylic acid. Thus, the RNA molecule can act as an RNA polymerase, differing from the protein enzyme in that it uses an internal rather than an external template. At pH 9, the same RNA enzyme has activity as a sequence-specific ribonuclease.
516 citations
TL;DR: The VAI RNA of adenovirus is a small, RNA polymerase III-transcribed species required for efficient translation of host cell and viral mRNAs late after infection and can be reproduced in extracts of interferon-treated cells.
347 citations
TL;DR: The ability to make infectious poliov virus RNA efficiently from cloned DNA makes it possible to apply techniques of in vitro mutagenesis to the analysis of poliovirus functions and the construction of novel and perhaps useful derivatives of pol Giovirus.
Abstract: Plasmids containing the entire cDNA sequence of poliovirus type 1 (Mahoney strain) under control of a promoter for T7 RNA polymerase have been constructed. Purified T7 RNA polymerase efficiently transcribes the entire poliovirus cDNA in either direction to produce full-length poliovirus RNA [(+)RNA] or its complement [(-)RNA]. The (+)RNA produced initially had 60 nucleotides on the 5' side of the poliovirus RNA sequence, including a string of 18 consecutive guanine residues generated in the original cloning and an additional 626 nucleotides of pBR322 sequence beyond the poly(A) tract at the 3' end. Such RNA, while much more infectious than the plasmid DNA, is only about 0.1% as infectious as RNA isolated from the virus. Subsequently, a T7 promoter was placed only 2 base pairs ahead of the poliovirus sequence, so that T7 RNA polymerase synthesizes poliovirus RNA with only 2 additional guanine residues at the 5' end and no more than seven nucleotides past the poly(A) tract at the 3' end. Such RNA has much higher specific infectivity, about 5% that of RNA isolated from the virus. The ability to make infectious poliovirus RNA efficiently from cloned DNA makes it possible to apply techniques of in vitro mutagenesis to the analysis of poliovirus functions and the construction of novel and perhaps useful derivatives of poliovirus. A source of variant RNAs should also allow detailed study of the synthesis and processing of poliovirus proteins in vitro.
339 citations
TL;DR: It is hypothesized that RNA polymerase II has access to, and a high affinity for, the promoter region of this gene before induction, and this poised RNA polymerases II may be critical in the mechanism of transcription activation.
Abstract: By using a protein-DNA cross-linking method (D. S. Gilmour and J. T. Lis, Mol. Cell. Biol. 5:2009-2018, 1985), we examined the in vivo distribution of RNA polymerase II on the hsp70 heat shock gene in Drosophila melanogaster Schneider line 2 cells. In heat shock-induced cells, a high level of RNA polymerase II was detected on the entire gene, while in noninduced cells, the RNA polymerase II was confined to the 5' end of the hsp70 gene, predominantly between nucleotides -12 and +65 relative to the start of transcription. This association of RNA polymerase II was apparent whether the cross-linking was performed by a 10-min UV irradiation of chilled cells with mercury vapor lamps or by a 40-microsecond irradiation of cells with a high-energy xenon flash lamp. We hypothesize that RNA polymerase II has access to, and a high affinity for, the promoter region of this gene before induction, and this poised RNA polymerase II may be critical in the mechanism of transcription activation.
338 citations
TL;DR: The hepatitis delta virus can be found in the serum and liver of some hepatitis B virus patients as mentioned in this paper, and the RNA genome of serum-derived delta virus is single-stranded and circular.
Abstract: 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.
309 citations
TL;DR: Evidence that addition of the 35 nucleotide spliced leader (SL) to the 5' end of T. brucei mRNAs occurs via trans RNA splicing is presented, and the 100-mer RNA is revealed by S1 nuclease analysis of total and poly(A)+ RNA.
298 citations
TL;DR: It is proposed that the RNA replication of MHV proceeds in a discontinuous and nonprocessive manner, thus generating free segmented RNA intermediates, which could be used in RNA recombination via a copy-choice mechanism.
Abstract: The RNA genome of coronaviruses consists of a single species of nonsegmented RNA. In this communication, we demonstrate that the RNA genomes of different strains of murine coronaviruses recombine during mixed infection at a very high frequency. Susceptible cells were coinfected with a temperature-sensitive mutant of one strain of mouse hepatitis virus (MHV) and a wild-type virus of a different strain. Of 21 randomly isolated viruses released from the coinfected cells at the nonpermissive temperature, 2 were recombinants which differed in the site of recombination. After three serial passages of the original virus pool derived from the mixed infection, the majority of the progeny viruses were recombinants. These recombinant viruses represented at least five different recombination sites between the two parental MHV strains. Such a high-frequency recombination between nonsegmented RNA genomes of MHV suggests that segmented RNA intermediates might be generated during MHV replication. We propose that the RNA replication of MHV proceeds in a discontinuous and nonprocessive manner, thus generating free segmented RNA intermediates, which could be used in RNA recombination via a copy-choice mechanism.
284 citations
TL;DR: It is established that, in contrast to the polymerase II-directed transcription of mammalian genes for U1-U5 small nuclear RNAs, human U6 RNA is transcribed by RNA polymerase III.
Abstract: A DNA fragment homologous to U6 small nuclear RNA was isolated from a human genomic library and sequenced. The immediate 5'-flanking region of the U6 DNA clone had significant homology with a potential mouse U6 gene, including a "TATA box" at a position 26-29 nucleotides upstream from the transcription start site. Although this sequence element is characteristic of RNA polymerase II promoters, the U6 gene also contained a polymerase III "box A" intragenic control region and a typical run of five thymines at the 3' terminus (noncoding strand). The human U6 DNA clone was accurately transcribed in a HeLa cell S100 extract lacking polymerase II activity. U6 RNA transcription in the S100 extract was resistant to alpha-amanitin at 1 microgram/ml but was completely inhibited at 200 micrograms/ml. A comparison of fingerprints of the in vitro transcript and of U6 RNA synthesized in vivo revealed sequence congruence. U6 RNA synthesis in isolated HeLa cell nuclei also displayed low sensitivity to alpha-amanitin, in contrast to U1 and U2 RNA transcription, which was inhibited greater than 90% at 1 microgram/ml. In addition, U6 RNA synthesized in isolated nuclei was efficiently immunoprecipitated by an antibody against the La antigen, a protein known to bind most other RNA polymerase III transcripts. These results establish that, in contrast to the polymerase II-directed transcription of mammalian genes for U1-U5 small nuclear RNAs, human U6 RNA is transcribed by RNA polymerase III.
TL;DR: A WNV mutant, which replicates progeny genome RNA more efficiently than parental WNV, was found to have a 3'-genomic sequence identical to that of its parent virus, indicating that this structure is important for the survival of the virus.
TL;DR: It seems possible that RNA catalysts might have played a part in prebiotic nucleic acid replication, prior to the availability of useful proteins, if the RNA enzyme could use another copy of itself as a template, RNA self-replication could be achieved.
Abstract: A shortened form of the self-splicing ribosomal RNA intervening sequence of Tetrahymena thermophila has enzymatic activity as a poly(cytidylic acid) polymerase [Zaug, A.J. & Cech, T.R. (1986) Science 231, 470-475]. Based on the known properties of this enzyme, a detailed model is developed for the template-dependent synthesis of RNA by an RNA polymerase itself made of RNA. The monomer units for RNA synthesis are tetra- and pentanucleotides of random base sequence. Polymerization occurs in a 5'-to-3' direction, and elongation rates are expected to approach two residues per minute. If the RNA enzyme could use another copy of itself as a template, RNA self-replication could be achieved. Thus, it seems possible that RNA catalysts might have played a part in prebiotic nucleic acid replication, prior to the availability of useful proteins.
Patent•
06 Mar 1986
TL;DR: In this article, a + strand RNA viral transformation of host organisms with foreign RNA, and expression of said foreign RNA was described, where the foreign RNA is inserted into an infective RNA viral segment containing replication elements, and allowed to infect the host organism.
Abstract: A + strand RNA viral transformation of host organisms with foreign RNA, and expression of said foreign RNA. The foreign RNA is inserted into an infective RNA viral segment containing replication elements, and allowed to infect the host organism. The invention is exemplified utilizing brome mosaic RNA modified to contain a gene codting for chloramphenicol acetyl transferase (CAT) in the transformation of barley protoplasts.
TL;DR: Data from a series of deletions covering the entire DI genome show that only sequences in the 162 nucleotide region at the 5' terminus and in the 19 nucleotide regions at the 3' termini are specifically required for replication and packaging of these genomes.
TL;DR: The structure of the double-stranded replicative form of RNA isolated from brome mosaic virus-infected leaves was consistent with such a mechanism occurring in vivo, in that it lacked the 3'-terminal A found on virion RNAs.
TL;DR: Methods are described which allow direct quantitation and sequence analysis of base substitution levels at predetermined single nucleotide positions in cloned pools of an RNA virus genome or in its RNA transcripts in vitro.
Abstract: Methods are described which allow direct quantitation and sequence analysis of base substitution levels at predetermined single nucleotide positions in cloned pools of an RNA virus genome or in its RNA transcripts in vitro. Base substitution frequencies for vesicular stomatitis virus (VSV) at one highly conserved site examined were reproducible and extremely high, averaging between 10(-4) and 4 X 10(-4) substitutions per base incorporated at this single site. If polymerase error frequencies averaged as high at all other sites in the 11-kilobase VSV genome, then every member of a cloned VSV population would differ from most other genomes in that clone at a number of different nucleotide positions. The preservation of a consensus sequence in such variable RNA virus genomes then could only result from strong biological selection (in a single host or multihost environment) for the most fit and competitive representatives of extremely heterogeneous virus populations.
TL;DR: The results suggest that the pregenome also donates a capped oligoribonucleotide that acts as the primer of plus-strand DNA synthesis, using the minus-strander DNA as template.
Abstract: The plus strand of virion DNA of duck hepatitis B virus possessed, at its 5' terminus, a capped oligoribonucleotide 18 to 19 bases in length. This oligoribonucleotide had a unique 5' end, the heterogeneity in length reflecting two distinct junctions with plus-strand DNA that were 1 base apart. The sequence of the RNA differed from that predicted by the sequence of duck hepatitis B virus upstream of the 5' ends of plus-strand DNA but was identical to a downstream sequence corresponding to the 5' terminus of a major poly(A)+ viral RNA mapped by Buscher and co-workers (Cell 40:717-724, 1985). This RNA transcript is thought to serve as the template (i.e., the pregenome) for minus-strand synthesis via reverse transcription. The results suggest that the pregenome also donates a capped oligoribonucleotide that acts as the primer of plus-strand DNA synthesis, using the minus-strand DNA as template.
TL;DR: In all specified regions, the vaccinia large subunit has greater homology with eukaryotic RNA polymerases II and III than with the E. coli polymerase.
Abstract: We have determined the nucleotide sequence of a region of the vaccinia virus genome encoding RNA polymerase subunits of 22 and 147 kDa and have mapped the 5' and 3' ends of the two mRNAs. The predicted amino acid sequence of the vaccinia 147-kDa subunit shows extensive homology with the largest subunit of Escherichia coli RNA polymerase, yeast RNA polymerases II and III, and Drosophila RNA polymerase II. The regions of homology between the five RNA polymerases are subdivided into five separate domains that span most of the length of each. A sixth domain shared by the vaccinia and the eukaryotic polymerases is absent from the E. coli sequence. In all specified regions, the vaccinia large subunit has greater homology with eukaryotic RNA polymerases II and III than with the E. coli polymerase. Vaccinia virus and eukaryotic RNA polymerases may therefore have evolved from a common ancestral gene after the latter diverged from prokaryotes.
TL;DR: The complex of T7 RNA polymerase with the phage phi 10 promoter has been visualized indirectly by exploiting the ability of the polymerase to protect DNA sequences from cleavage by methidiumpropyl-EDTA X Fe(II).
Abstract: Promoters for T7 RNA polymerase have a highly conserved sequence of 23 continuous base pairs located at position -17 to +6 relative to the initiation site for the RNA. The complex of T7 RNA polymerase with the phage phi 10 promoter has been visualized indirectly by exploiting the ability of the polymerase to protect DNA sequences from cleavage by methidiumpropyl-EDTA X Fe(II). The DNA contacts made by T7 RNA polymerase have been mapped during binding and during the subsequent initiation of transcription. The RNA polymerase alone protects 19 bases in a region from -21 to -3. Synthesis of the trinucleotide r(GGG) expands the length of the sequence protected by the RNA polymerase and stabilizes the complex; 29 bases (-21 to +8) are protected, and the observed equilibrium association constant of the r(GGG) complex is 5 X 10(5) M-1. The formation of a hexanucleotide mRNA, r(GGGAGA), further extends the protected region; 32 bases (-21 to +11) are protected. Finally, the synthesis of a pentadecanucleotide mRNA leads to a translocation of the region protected by the protein; the sequence now protected is reduced to 24 bases (-4 to +20).
TL;DR: The ColE1 primer transcript (RNA II) hybridizes to the template DNA near the replication origin and is cleaved by RNAase H to form the primer, which is inhibited by binding of another RNA, RNA I, to RNA II.
TL;DR: It is shown here that transcripts of full-length and truncated DNA clones of the satellite RNA sequence also process in a nonenzymic reaction, and one such transcript was an RNA that has about one-fourth of theatellite RNA sequence, representing the 3'-terminal and 5-terminal portions of monomeric RNA joined in the junction that is cleaved in dimeric RNA.
Abstract: The satellite RNA of tobacco ringspot virus depends upon tobacco ringspot virus for its replication and source of coat protein. The satellite RNA reduces virus accumulation and the severity of virus-induced symptoms. Repetitive sequence, dimeric, and higher forms of the satellite RNA are known to autolytically process to form biologically active monomeric RNA of 359 nucleotide residues [Prody, G. A., Bakos, J. T., Buzayan, J. M., Schneider, I. R. & Bruening, G. (1986) Science 231, 1577-1580], with a 5'-hydroxyl and a 2',3'-cyclic phosphodiester as the new terminal groups. We show here that transcripts of full-length and truncated DNA clones of the satellite RNA sequence also process in a nonenzymic reaction. One such transcript was an RNA that has about one-fourth of the satellite RNA sequence, representing the 3'-terminal and 5'-terminal portions of monomeric RNA joined in the junction that is cleaved in dimeric RNA. This RNA autolytically processed more efficiently than molecules with a larger proportion of the satellite RNA nucleotide sequence.
TL;DR: The insensitivity to RNA I of transcripts longer than 360 nucleotides is not due to the absence of RNA I binding but to this binding having no effect on primer formation, which is crucial to regulation.
TL;DR: Conditions are described for programming with active transcription complexes essentially all the Xenopus 5S RNA genes added to an extract of Xenopus oocyte nuclei, calling this rate enhancement rate enhancement.
TL;DR: The temporal sequence of coronavirus plus and minus-strand RNA synthesis was determined in 17CL1 cells infected with the A59 strain of mouse hepatitis virus and the synthesis of both genome and subgenomic mRNAs and of viral minus strands required continued protein synthesis.
Abstract: The temporal sequence of coronavirus plus-strand and minus-strand RNA synthesis was determined in 17CL1 cells infected with the A59 strain of mouse hepatitis virus (MHV). MHV-induced fusion was prevented by keeping the pH of the medium below pH 6.8. This had no effect on the MHV replication cycle, but gave 5- to 10-fold-greater titers of infectious virus and delayed the detachment of cells from the monolayer which permitted viral RNA synthesis to be studied conveniently until at least 10 h postinfection. Seven species of poly(A)-containing viral RNAs were synthesized at early and late times after infection, in nonequal but constant ratios. MHV minus-strand RNA synthesis was first detected at about 3 h after infection and was found exclusively in the viral replicative intermediates and was not detected in 60S single-stranded form in infected cells. Early in the replication cycle, from 45 to 65% of the [3H]uridine pulse-labeled RF core of purified MHV replicative intermediates was in minus-strand RNA. The rate of minus-strand synthesis peaked at 5 to 6 h postinfection and then declined to about 20% of the maximum rate. The addition of cycloheximide before 3 h postinfection prevented viral RNA synthesis, whereas the addition of cycloheximide after viral RNA synthesis had begun resulted in the inhibition of viral RNA synthesis. The synthesis of both genome and subgenomic mRNAs and of viral minus strands required continued protein synthesis, and minus-strand RNA synthesis was three- to fourfold more sensitive to inhibition by cycloheximide than was plus-strand synthesis.
TL;DR: This work constructed several well-defined mutations in the nonstructural portion of the poliovirus type I (Mahoney strain) genome by making small insertions in an infectious cDNA clone, providing the first unambiguous evidence that the non structures of thePoliovirus genome contains multiple complementation groups.
Abstract: We constructed several well-defined mutations in the nonstructural portion of the poliovirus type I (Mahoney strain) genome by making small insertions in an infectious cDNA clone. The derived viral strains carrying the mutations exhibited a variety of distinct plaque phenotypes. Thus, we were able to examine genetic complementation between different pairs of mutants by comparing the yields of progeny virus in mixed and single infections. Two mutants bearing lesions in the 2A and 3A regions of the genome, which are defective in the inhibition of host cell translation and the synthesis of viral RNA, respectively, could be rescued efficiently by genetic complementation; three replication-deficient mutants containing insertions in the 2B, 3D (replicase), and 3'-untranslated regions could not. Both the 2A and 3A mutants could be rescued by each other and by all of the other mutants tested. Because yield enhancement was apparent well before the completion of a single infectious cycle, it is likely that complementation of both mutants involved early diffusion of functional products. These data provide the first unambiguous evidence that the nonstructural portion of the poliovirus genome contains multiple complementation groups. The data also suggest that certain nonstructural functions act only in cis.
TL;DR: The net ligation reaction of STobRV (-)RNA is the precise reversal of autolysis, and its implications for the formation of multimeric STOBRV RNAs during replication are discussed.
Abstract: The satellite RNA of tobacco ringspot virus (STobRV RNA) replicates and becomes encapsidated in association with tobacco ringspot virus. Previous results show that the infected tissue produces multimeric STobRV RNAs of both polarities. RNA that is complementary to encapsidated STobRV RNA, designated as having the (-) polarity, cleaves autolytically at a specific ApG bond. Purified autolysis products spontaneously join in a non-enzymic reaction. We report characteristics of this RNA ligation reaction: the terminal groups that react, the type of bond in the newly formed junction and the nucleotide sequence of the joined RNA. The nucleotide sequence of the ligated RNA shows that joining of the reacting RNAs restored an ApG bond. The junction ApG has a 3'-to-5' phosphodiester bond. Thus the net ligation reaction of STobRV (-)RNA is the precise reversal of autolysis. We discuss this new type of RNA ligation reaction and its implications for the formation of multimeric STobRV RNAs during replication.
TL;DR: The chiral stationary phase for double-stranded RNA replication showed good chiral recognition ability for dsRNAs of both Toxin-Immunity Precursor Protein and Viruslike Particle Coat Protein levels.
Abstract: PERSPECTIVES AND SUMMARY 373 DOUBLE-STRANDED RNAs IN SACCHAROMYCES CEREVlSIAE ........ 375 M dsRNAs: The Toxin-Immunity Precursor Protein . 375 The L-A Family: [HOK], [NEX], [EXL], and Viruslike Particle Coat Protein . ... . .. 381 L-B and L-C dsRNA 382 T and W dsRNAs.. . . . . . . . . ....... .. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . ... . . . . .. . . .. .. . . . . . . . . . . . ...... 383 TRANSCRIPTION OF DOUBLE-STRANDED RNA 383 REPLICATION OF DOUBLE-STRANDED RNA 384 REGULATION OF DOUBLE-STRANDED RNA REPLICATION . . . . .. . . . .. . . . . . . . . . . . . . . 388 CONCLUDING REMARKS . 391
TL;DR: It is demonstrated that, during a mixed infection with two different MHVs, the leader RNA sequences from one virus could be detected on the m RNAs of the coinfecting virus at a high frequency, as if the leader sequence and mRNAs were joined together from two randomly segregating RNA segments.
Abstract: Mouse hepatitis virus (MHV), which replicates in cytoplasm of infected cells, contains an identical leader RNA sequence at the 5' end of each of the virus-specific mRNAs. Previous studies suggested that the synthesis of these mRNAs does not involve conventional RNA splicing and may instead require priming by a free leader RNA. In this communication, we demonstrate that, during a mixed infection with two different MHVs, the leader RNA sequences from one virus could be detected on the mRNAs of the coinfecting virus at a high frequency, as if the leader sequence and mRNAs were joined together from two randomly segregating RNA segments. This finding demonstrates that MHV mRNA transcription utilizes independently transcribed leader RNA species that possess the trans-acting property. This study thus provides further evidence in support of the unique model of "leader-primed transcription" for coronavirus mRNA synthesis.