Showing papers on "RNA-dependent RNA polymerase published in 1984"

Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter

Abstract: A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).

Regulation of ribosomal RNA promoters with a synthetic lac operator

Abstract: A synthetic 21-base-pair long DNA fragment containing the central lac operator sequence has been inserted near the initiation point of the cloned Escherichia coli rrnB rRNA promoter P2 in the natural and reverse orientation. RNA synthesis is efficiently repressed in both orientations in lac Iq strains and is induced with isopropyl beta-D-thiogalactoside. When the rrnB promoter P1 is also present, upstream from P2 and the synthetic lac operator, repression of transcription is incomplete. The levels of transcription were measured in vivo, indirectly by the expression of a protein (chloramphenicol acetyltransferase), or directly by the expression of a stable RNA (E. coli 4.5S RNA) in a simple assay involving gel electrophoresis of unlabeled total RNA from E. coli. The rrnB promoter constructions can produce high levels of protein expression as well as high levels of expression of stable RNA.

Specific interaction between the self-splicing RNA of Tetrahymena and its guanosine substrate: implications for biological catalysis by RNA

Abstract: Splicing of the ribosomal RNA precursor of Tetrahymena has previously been shown to require no protein in vitro; the cleavage-ligation activity is intrinsic to the RNA molecule. Analysis of the reaction kinetics with guanosine, which is a substrate in the reaction, and with several guanosine analogues suggests that guanosine binds to a specific site on the pre-rRNA. It appears that the RNA, like an enzyme, binds its substrate to promote the rate and specificity of a biological reaction.

Catalytic activity of an RNA molecule prepared by transcription in vitro

Abstract: Ribonuclease P is a ribonucleoprotein that cleaves precursors to transfer RNA (tRNA) molecules to yield the correct 5' terminal sequences of the mature tRNA's. The RNA moiety M1 RNA of ribonuclease P from Escherichia coli and the unprocessed transcript prepared in vitro of the gene for M1 RNA can both perform the cleavage reactions of the canonical enzyme in the absence of the protein moiety. When the transcript of the M1 RNA gene is combined with the protein moiety not only is a tRNA precursor cleaved but also the precursor to 4.5S RNA from Escherichia coli.

Detecting protein-DNA interactions in vivo: distribution of RNA polymerase on specific bacterial genes.

Abstract: We present an approach for determining the in vivo distribution of a protein on specific segments of chromosomal DNA. First, proteins are joined covalently to DNA by irradiating intact cells with UV light. Second, these cells are disrupted in detergent, and a specific protein is immunoprecipitated from the lysate. Third, the DNA that is covalently attached to the protein in the precipitate is purified and assayed by hybridization. To test this approach, we examine the cross-linking in Escherichia coli of RNA polymerase to a constitutively expressed, lambda cI gene, and to the uninduced and isopropyl beta-D-thiogalactoside (IPTG)-induced lac operon. As expected, the recovery of the constitutively expressed gene in the immunoprecipitate is dependent on the irradiation of cells and on the addition of RNA polymerase antiserum. The recovery of the lac operon DNA also requires transcriptional activation with IPTG prior to the cross-linking step. After these initial tests, we examine the distribution of RNA polymerase on the leucine operon of Salmonella in wild-type, attenuator mutant, and promoter mutant strains. Our in vivo data are in complete agreement with the predictions of the attenuation model of regulation. From these and other experiments, we discuss the resolution, sensitivity, and generality of these methods.

Striking similarities in amino acid sequence among nonstructural proteins encoded by RNA viruses that have dissimilar genomic organization

Abstract: The plant viruses alfalfa mosaic virus (AMV) and brome mosaic virus (BMV) each divide their genetic information among three RNAs while tobacco mosaic virus (TMV) contains a single genomic RNA. Amino acid sequence comparisons suggest that the single proteins encoded by AMV RNA 1 and BMV RNA 1 and by AMV RNA 2 and BMV RNA 2 are related to the NH2-terminal two-thirds and the COOH-terminal one-third, respectively, of the largest protein encoded by TMV. Separating these two domains in the TMV RNA sequence is an amber termination codon, whose partial suppression allows translation of the downstream domain. Many of the residues that the TMV read-through domain and the segmented plant viruses have in common are also conserved in a read-through domain found in the nonstructural polyprotein of the animal alphaviruses Sindbis and Middelburg. We suggest that, despite substantial differences in gene organization and expression, all of these viruses use related proteins for common functions in RNA replication. Reassortment of functional modules of coding and regulatory sequence from preexisting viral or cellular sources, perhaps via RNA recombination, may be an important mechanism in RNA virus evolution.

Autocatalytic replication of recombinant RNA

Abstract: This invention concerns recombinant RNA molecules comprising a recognition sequence for the binding of an RNA-directed RNA polymerase, a sequence for the initiation of product strand synthesis and a heterologous sequence of interest inserted at a specific site in the internal region of the recombinant molecule. Such recombinant RNA molecules are capable of serving as a template for the synthesis of complementary single-stranded molecules by RNA-directed RNA polymerase. The product molecules so formed are also capable of serving as a template for the synthesis of additional copies of the original recombinant RNA molecule. In a preferred embodiment of the invention Qβ replicase is used as the RNA-directed RNA polymerase.

Characterization of leader RNA sequences on the virion and mRNAs of mouse hepatitis virus, a cytoplasmic RNA virus

Abstract: Mouse hepatitis virus, which replicates in cytoplasm, contains leader RNA sequences at the 5' end of the virus-specific mRNAs. We have sequenced this leader RNA by synthesizing cDNA from a synthetic oligodeoxyribonucleotide primer (15-mer) that is complementary to the sequences at the junction site between the leader and body sequences of the mRNAs. The leader sequences on each mRNA have exactly the same size, which span approximately equal to 70 nucleotides. Leader cDNA fragments obtained from several mRNA species were sequenced and found to be identical. Computer analysis of the leader RNA sequences shows that they share extensive sequence homology with the long-terminal-repeat region of several mammalian sarcoma viruses, suggesting possible common functions. This is a novel case of spliced leader sequences in the mRNAs of a cytoplasmic virus. An identical leader sequence is also present at the 5' end of the virion genomic RNA. The leader RNA is thus probably encoded by the virion genomic RNA template and is fused to the different body sequences of the various mRNAs. Since conventional RNA splicing is not involved, a novel mechanism for fusing two noncontiguous RNA segments in the cytoplasm must be utilized during viral transcription. Several minor cDNA bands longer than the leader were also synthesized, suggesting the possible presence of partially homologous sequences in other parts of the genome RNA.

Plant Viral Double-Stranded RNA

Abstract: Plant virologists can use several approaches to detect and diagnose viruses in plants. The menu of alternates has been examined critically by Hamilton et al (48). It includes attention to symptoms, host range, in vitro properties of the infectious agent, vector relationships and other aspects of transmissibility, particle morphology, the physical and chemical properties of the virus particle, its capsid protein and genome, and serological tests in many forms. The physical detection of viroid nucleic acids and the power of nucleic acid hybridization, cloning, and sequencing have been introduced (44, 80), and these methods will become important in the future. The available methods for the detection and diagnosis of viruses serve us well and will continue to do so. One purpose of this article is to review another tool available to the diagnostician. This is the detection and description of viral double-stranded (ds) RNA, and the use of the purified dsRNA as a reagent or an inoculum. The dsRNA approach is based on the premise that plants not infected with RNA viruses or virus-like agents do not contain readily detectable amounts of

Sequencing studies of pichinde arenavirus S RNA indicate a novel coding strategy, an ambisense viral S RNA.

Abstract: Analyses of the complete sequence of the 1.1 X 10(6)-dalton, small (S) RNA of the arenavirus Pichinde and virus-induced cellular RNA species have revealed that the viral nucleoprotein, N, is coded in a subgenomic, non-polyadenylated, virus-complementary mRNA corresponding to the 3' half of the viral RNA (Auperin et al., Virology 134:208-219, 1984). By contrast, a second S-coded product, presumably the viral glycoprotein precursor (GPC), is coded in a subgenomic, virus-sense mRNA corresponding to the 5' half of the RNA. Between the two genes is a unique RNA sequence that can be arranged in a hairpin configuration and may function as a transcription terminator for both genes. The term ambisense RNA is coined to describe this novel coding strategy of a viral RNA. The unique feature of the strategy is that the presumptive GPC mRNA and its translation product cannot be made until viral RNA replication has commenced. In addition, it allows the two subgenomic mRNA species to be regulated independently from each other or from other viral mRNA species. The implications of this strategy on possible mechanisms for the induction and maintenance of viral persistence, an important attribute of arenavirus infections, are discussed.

UAG readthrough during TMV RNA translation: isolation and sequence of two tRNAsTyr with suppressor activity from tobacco plants

Abstract: The hypothetical replicase or replicase subunit cistron in the 5'-proximal part of tobacco mosaic virus (TMV) RNA yields a major 126-K protein and a minor 183-K ;readthrough' protein in vivo and in vitro. Two natural suppressor tRNAs were purified from uninfected tobacco plants on the basis of their ability to promote readthrough over the corresponding UAG termination codon in vitro. In a reticulocyte lysate the yield of 183-K readthrough protein increases from 10% in the absence of added tobacco plant tRNA up to 35% in the case of pure tRNA added. Their amino acid acceptance and anticodon sequence (GpsiA) identifies the two natural suppressor tRNAs as the two normal major cytoplasmic tyrosine-specific tRNAs. tRNA(1) has an A:U pair at the base of the TpsiC stem and an unmodified G(10), whereas tRNA(2) contains a G:C pair in the corresponding location and mG in position 10. This is the first case that, in a higher eukaryote, the complete structure is known of both the natural suppressor tRNAs and the corresponding viral RNA on which they exert their function. The corresponding codon-anticodon interaction, which is not in accordance with the wobble hypothesis, and the possible biological significance of the readthrough phenomenon is discussed.

N protein alone satisfies the requirement for protein synthesis during RNA replication of vesicular stomatitis virus.

Abstract: Genomic replication of the negative-strand RNA viruses is dependent upon protein synthesis. To examine the requirement for protein synthesis in replication, we developed an in vitro system that supports the genome replication of defective interfering particles of the negative-strand rhabdovirus vesicular stomatitis virus (VSV), as a function of protein synthesis (Wertz, J. Virol. 46:513-522, 1983). The system consists of defective interfering nucleocapsid templates and an mRNA-dependent reticulocyte lysate to support protein synthesis. We report here an analysis of the requirement for individual viral proteins in VSV replication. Viral mRNAs purified by hybridization to cDNA clones were used to direct the synthesis of individual proteins in the in vitro system. By this method, it was demonstrated that the synthesis of the VSV nucleocapsid protein, N, alone, resulted in the replication of genome-length RNA by both defective interfering intracellular nucleocapsids and virion-derived nucleocapsids. Neither the viral phosphoprotein, NS, nor the matrix protein, M, supported RNA replication. The amount of RNA replication for a given amount of N protein was the same in reactions in which either all of the VSV proteins or only N protein were synthesized. In addition, RNA replication products synthesized in reactions containing only newly made N protein assembled with the N protein to form nucleocapsids. These results demonstrate that the major nucleocapsid protein (N) can by itself fulfill the requirement for protein synthesis in RNA replication and allow complete replication, i.e., initiation and elongation, as well as encapsidation of genome-length progeny RNA.

Homologous sequences in non-structural proteins from cowpea mosaic virus and picornaviruses.

Abstract: Computer analyses have revealed sequence homology between two non-structural proteins encoded by cowpea mosaic virus (CPMV), and corresponding proteins encoded by two picornaviruses, poliovirus and foot-and-mouth disease virus. A region of 535 amino acids in the 87-K polypeptide from CPMV was found to be homologous to the RNA-dependent RNA polymerases from both picornaviruses, the best matches being found where the picornaviral proteins most resemble each other. Additionally, the 58-K polypeptide from CPMV and polypeptide P2-X from poliovirus contain a conserved region of 143 amino acids. Based on the homology observed, a genetic map of the CPMV genome has been constructed in which the 87-K polypeptide represents the core polymerase domain of the CPMV replicase. These results have implications for the evolution of RNA viruses, and mechanisms are discussed which may explain the existence of homology between picornaviruses (animal viruses with single genomic RNAs) and comoviruses (plant viruses with two genomic RNAs).

The cDNA sequences of the sea urchin U7 small nuclear RNA suggest specific contacts between histone mRNA precursor and U7 RNA during RNA processing.

Abstract: 3' Processing of sea urchin H3 histone pre-mRNA depends on a small nuclear RNP which contains an RNA of nominally 60 nucleotide length, referred to below as U7 RNA. The U7 RNA can be enriched by precipitation of sea urchin U-snRNPs with human systematic lupus erythematosus antiserum of the Sm serotype. We have prepared cDNA clones of U7 RNA and determined by hybridization techniques that this RNA is present in sea urchin eggs at 30-fold lower molar concentration than U1 RNA. The RNA sequences derived from an analysis of eight U7 cDNA clones show neither homologies nor complementarities to any other know U-RNAs. The 3' portion of the presumptive RNA sequence can be folded into a stem-loop structure. The 5'-terminal sequences would be largely unstructured as free RNA. Their most striking feature is their base complementarity to the 3' conserved sequences of histone pre-mRNAs. Six out of nine bases of the conserved CAAGAAAGA sequence of the histone mRNA precursor and 13 out of 16 nucleotides from the conserved palindrome can be base paired with presumptive U7 RNA sequence, suggesting a unique hybrid structure for a processing intermediate formed from histone precursor and U7 RNA.

La Crosse virions contain a primer-stimulated RNA polymerase and a methylated cap-dependent endonuclease.

Abstract: Purified La Crosse virions in vitro were found to transcribe their negative polarity (-)RNA genomes. This polymerase activity was stimulated by oligonucleotides such as (A)nG, cap analogs such as m7GpppAm, and natural mRNAs such as alfalfa mosaic virus RNA 4. For (A)nG- and alfalfa mosaic virus RNA 4-stimulated reactions, evidence is presented that these RNAs stimulate activity by acting as primers for viral transcription. The cap analogs appear to stimulate activity via an alternative mechanism. Purified La Crosse virions were also found to contain an endonuclease which specifically cleaves alfalfa mosaic virus RNA 4 when this RNA contains a methylated cap group.

Identifier sequences are transcribed specifically in brain

Abstract: 'Identifier' or ID sequences are present in 62% of the RNA polymerase II and III transcripts made in vitro from brain nuclei but in fewer than 4% of the transcripts made from the nuclei of other tissues. An homologous 160-nucleotide cytoplasmic poly(A)+ RNA species, BC1, and a smaller species, BC2, are located in vivo exclusively in neural tissues. Cloned ID sequences are polymerase III templates in vitro. Our data suggest a model in which brain-specific polymerase III transcription of ID sequences located in introns of brain genes activates those genes in a primary manner for polymerase III transcription.

Mutant viral RNAs synthesized in vitro show altered aminoacylation and replicase template activities.

Abstract: A remarkable feature of the genomic RNAs of several plant viruses is the presence at the 3′ end of a region that exhibits tRNA-like functions, including aminoacylation1–3. The three genomic and single subgenomic RNAs of brome mosaic virus (BMV) accept tyrosine in vitro4 and in vivo5, the smallest 3′ fragment that can be aminoacylated being about 135 nucleotides long6. The roles of the tRNA-like properties are incompletely understood, but an involvement in replication rather than translational functions is likely3,7,8. We have recently shown (J.J.B. et al., in preparation) that the features recognized by the BMV RNA-specific RNA-dependent RNA polymerase (replicase)9,10 for the use of BMV RNA for complementary strand synthesis also lie within the tRNA-like structure. To distinguish between the roles of BMV RNA as a substrate for tyrosyl-tRNA synthetase and BMV replicase, we have now produced BMV RNAs with mutations at two separate loci within the tRNA-like structure. This has been achieved by transcription in vitro from specifically mutagenized cDNA, an approach permitting the generation of targeted mutants without regard to their viability in vivo.

Rubella virus 40S genome RNA specifies a 24S subgenomic mRNA that codes for a precursor to structural proteins.

Abstract: We have analyzed the structure of the rubella virus genome RNA and the virus-specific RNA species synthesized in B-Vero cells infected with rubella virus. A single-stranded, capped, and polyadenylated RNA species sedimenting at 40S in a sucrose gradient was released from purified virions treated with sodium dodecyl sulfate. This RNA species migrated with an Mr of about 3.8 X 10(6) in an agarose gel after denaturation with glyoxal and dimethyl sulfoxide. Infected cells labeled with [3H]uridine in the presence of actinomycin D contained, in addition to the 40S RNA, a single-stranded polyadenylated 24S RNA species as shown by sucrose gradient analysis. In a Northern blot analysis, this RNA hybridized to a cDNA probe derived from the 3' portion of the genomic 40S RNA. In vitro translation of the 24S RNA species yielded a 110,000-dalton polypeptide, in addition to some smaller products which were immunoprecipitated with an antiserum prepared against the structural proteins E1, E2a, E2b, and C. Since the sum of the molecular weights of the nonglycosylated envelope proteins and the capsid protein has been estimated to be about 116,000 (C. Oker-Blom et al., J. Virol. 46:964-973, 1983), these results suggest that the 24S RNA species represents a subgenomic mRNA coding for a precursor (p110) to the structural proteins of rubella virus. Thus, the strategy of gene expression of rubella virus appears to be similar to that of the alphaviruses.

Temporal regulation of murine cytomegalovirus transcription and mapping of viral RNA synthesized at immediate early times after infection.

Abstract: The replication of murine cytomegalovirus strain Smith in murine embryonic fibroblasts was investigated at immediate early, early, and late times after infection. Cloned subgenomic HindIII fragments of murine cytomegalovirus DNA served to define the regions of transcription. At immediate early times viral RNA classes ranging in size from 5.1 to 1.05 kilobases (kb) were transcribed mainly from the fragments HindIII-K and -L, whereas low levels of transcription were detected from the two termini HindIII-E and HindIII-N. A characteristic pattern of proteins could be translated from immediate early RNA in vitro. At early and late times after infection transcription from all HindIII fragments occurred, but different patterns of transcripts and proteins could be identified. Inhibitors of DNA synthesis induced differences in the late transcription pattern, located in the HindIII-F fragment. The coding region for abundant immediate early transcription could be located at between 0.769 and 0.817 map units. A plasmic clone containing the main part (0.769 to 0.815 map units) of this region was constructed. This region coded for six polyadenylated immediate early RNA species of 5.1, 2.75, 2.0, 1.75, 1.65, and 1.05 kb in size. Only the 1.75-kb RNA originated entirely from the HindIII-L fragment. The 5.1- and 2.75-kb RNA species were encoded by both the HindIII-L and HindIII-K fragments, and the 2.0-, 1.65-, and 1.05-kb RNA species were entirely transcribed within HindIII-K.

La antigen recognizes and binds to the 3'-oligouridylate tail of a small RNA.

Abstract: The La antigen is a cellular protein which interacts with many RNA species that are products of RNA polymerase III, including the adenovirus virus-associated (VA) RNAs. We demonstrate that the efficiency of antigen binding in vitro is determined by the number of U residues at the RNA 3' terminus. Forms of VA RNAI with more than two terminal U residues are fully bound, forms with two U residues are partially bound, and forms with fewer than two U residues are not bound at all. The antigen can be covalently linked to VA RNA by UV irradiation, and the site of cross-linking is shown to contain the 3' terminus of the RNA. We conclude that the antigen recognizes the U-rich 3' tail of VA RNA, and presumably that of other polymerase III products, and that it binds at or close to this site.

Promoter selectivity of Escherichia coli RNA polymerase. II: Altered promoter selection by mutant holoenzymes.

Abstract: Using the in vitro mixed transcription system (Kajitani and Ishihama (1983a, 1983b), we examined selective transcription of truncated DNA templates carrying lac(UV5), rrnE or rpsA promoters by RNA polymerase holoenzymes from pairs of wild-type parents and mutants with a mutation in one or more RNA polymerase subunit genes. The promoter selectivity of RNA polymerases from two sigma-subunit mutants carrying either rpoD2 or rpoD285 differed markedly from that of the respective wild-type enzymes. Both the parental RNA polymerases, however, exhibited abnormal promoter selectivity compared with holoenzymes from various wild-type E. coli strains. On the other hand, all the RNA polymerases from rpoB and/or rpoC mutants and the respective wild-type parents were similar, if not identical, in promoter selection at low temperature. At high temperature, however, RNA polymerases from mutants carrying rpoB2B7 and rpoC4, affecting the beta and beta' subunits, respectively, showed decreased transcription from the high-affinity slow-transcribable promoter rrnEp2 whereas the rpoC92 and rpoB906 X rpoC907 mutant enzymes both lost transcription activity from the strong promoter lacP(UV5). Taking all these observations together we conclude that not only the sigma subunit but also the beta and beta' subunits are involved in the recognition of promoters.