Showing papers on "RNA-dependent RNA polymerase published in 1988"
TL;DR: In vitro mutagenesis of sequences required for the self-catalysed cleavage of a plant virus satellite RNA has allowed definition of an RNA segment with endoribonuclease activity.
Abstract: In vitro mutagenesis of sequences required for the self-catalysed cleavage of a plant virus satellite RNA has allowed definition of an RNA segment with endoribonuclease activity. General rules have been deduced for the design of new RNA enzymes capable of highly specific RNA cleavage, and have been successfully tested against a new target sequence.
1,383 citations
TL;DR: It is shown that a promoter-associated RNA polymerase II molecule is transcriptionally engaged and has formed a nascent RNA chain, but is apparently arrested at that point and unable to penetrate further into the hsp70 gene without heat induction.
631 citations
565 citations
TL;DR: It is shown that the RNA replicase of Semliki Forest and Sindbis virus is located in complex ribonucleoprotein structures associated with the cytoplasmic surface of modified secondary lysosomes and endosomes.
Abstract: Using morphological and cell biological techniques, we have shown that the RNA replicase of Semliki Forest and Sindbis virus (two closely related alphaviruses) is located in complex ribonucleoprotein structures associated with the cytoplasmic surface of modified secondary lysosomes and endosomes. These nucleoprotein complexes often form a bridge between the membrane of the endocytic vacuole and the rough endoplasmic reticulum where the synthesis of the structural proteins of these viruses occurs. The results suggest that these cytopathic vacuoles constitute sites not only for viral RNA synthesis, but also for translation of structural proteins, and for the assembly of nucleocapsids.
379 citations
TL;DR: In vitro the RNA complementary to the HDV genome, the antigenomic RNA, could undergo a self-cleavage that was not only more than 90% efficient but also occurred only at a single location.
Abstract: The structure and replication of the single-stranded circular RNA genome of hepatitis delta virus (HDV) are unique relative to those of known animal viruses, and yet there are real similarities between HDV and certain infectious RNAs of plants. Therefore, since some of the latter RNAs have been shown to undergo in vitro site-specific cleavage and even ligation, we tested the hypothesis that similar events might also occur for HDV RNA. In partial confirmation of this hypothesis, we found that in vitro the RNA complementary to the HDV genome, the antigenomic RNA, could undergo a self-cleavage that was not only more than 90% efficient but also occurred only at a single location. This cleavage was found to produce junction fragments consistent with a 5'-hydroxyl and a cyclic 2',3'-monophosphate. Since the observed cleavage was both site-specific and occurred only once per genome length, we propose that the site may be relevant to the normal intracellular replication of the HDV genome. Because the site is located almost adjacent to the 3' end of the delta antigen-coding region, the only known functional open reading frame of HDV, we suggest that the cleavage may have a role not only in genome replication but also in RNA processing, helping to produce a functional mRNA for the translation of delta antigen.
360 citations
TL;DR: This article used oligonucleotide-directed mutagenesis to generate a series of mutations in the nucleocapsid protein-coding region of Moloney murine leukemia virus.
Abstract: All retroviruses encode a nucleic acid-binding or nucleocapsid protein that is believed to be associated with RNA in the virion. Further, all retroviral nucleocapsid proteins contain either one or two copies of the sequence Cys-Xaa2-Cys-Xaa4-His-Xaa4-Cys. The conservation of this sequence suggested that it is important for virus replication, and its resemblance to the "zinc-finger" sequences found in eukaryotic transcription factors raised the possibility that it recognizes specific sequences in viral RNA during retrovirus assembly. We used oligonucleotide-directed mutagenesis to generate a series of mutations in the nucleocapsid protein-coding region of Moloney murine leukemia virus. These mutations changed single amino acids, including each of the cysteines, to serine. The mutant viral genomes direct the synthesis of virus particles; these particles lack detectable viral RNA but do contain significant levels of cellular RNAs. Thus it appears that the mutations have destroyed the ability of the viral proteins to specifically package viral RNA during virus assembly. We propose that the conserved sequence in retroviral nucleocapsid proteins functions in RNA sequence recognition and suggest that it is evolutionarily related to the zinc fingers that recognize specific sequences in double-stranded DNA.
327 citations
TL;DR: The synthesized recombinant RNA molecules that function both as hybridization probes and as templates for exponential amplification by Qβ replicase demonstrate the feasibility of employing exponentially replicatable RNAs in bioassays, where they serve the dual role of specific probe and amplifiable reporter.
Abstract: We have synthesized recombinant RNA molecules that function both as hybridization probes and as templates for exponential amplification by Qβ replicase. Each recombinant consists of a sequence specific for the protozoan parasite, Plasmodium falciparum, embedded within the sequence of MDV-1 RNA, which is a natural template for Qβ replicase. The probe sequence was inserted within a hairpin loop that occurs on the exterior of MDV-1 RNA. The recombinant RNAs hybridize specifically to complementary DNA, despite topological constraints on the probe domain, are replicated at the same rate as MDV-1 RNA, despite their additional length, and are able to serve as templates for the synthesis of a large number of RNA copies. A Qβ replicase reaction initiated with only 0.14 femtograms of recombinant RNA (1,000 molecules) can produce 129 nanograms of recombinant RNA product in 30 minutes. This represents a one-billion fold amplification. Our results demonstrate the feasibility of employing exponentially replicatable RNAs in bioassays, where they would serve the dual role of specific probe and amplifiable reporter.
326 citations
TL;DR: It was determined that the minimum length of contiguous sequence needed for self-cleavage of genomic RNA was 30 bases 5' and 74 bases 3' of the cleavage site, which was not compatible with the "hammerhead" structure hypothesized to be important in the self- Cleavage reactions of other RNAs.
Abstract: Recently we reported that in vitro RNA transcripts complementary to the genome of hepatitis delta virus (HDV) contain a unique site at which self-cleavage can occur. Subsequent studies showed that a similar self-cleavage site was present on in vitro RNA transcripts of genomic HDV RNA. The same self-cleavage reactions were also found to occur on HDV RNAs from the livers of infected chimpanzees. Using the in vitro RNA it was also possible to determine that the minimum length of contiguous sequence needed for self-cleavage of genomic RNA was 30 bases 5' and 74 bases 3' of the cleavage site. This sequence was not compatible with the "hammerhead" structure hypothesized to be important in the self-cleavage reactions of other RNAs.
289 citations
TL;DR: Transcription activates the origin of an otherwise inert plasmid by altering the local DNA structure, facilitating its opening by dnaA protein during the assembly of replication forks.
221 citations
TL;DR: Results indicate that the first strand transfer step in reverse transcription is an intermolecular event while that of the second transfer is intramolecular, and retroviruses contain two functionally active RNA's, and both may be required for the generation of a single linear DNA molecule.
Abstract: Retroviruses contain two copies of the plus stranded viral RNA genome. As a means of determining whether both of these RNA's are used in the reverse transcription reaction, cells were infected with heterozygous virus particles that varied in nucleotide sequence at two separate locations at the RNA termini. The DNA proviruses formed from a single cycle of reverse transcription were then examined. Of the 12 proviruses that were characterized, all exhibited long terminal repeats (LTR's) that would be expected to arise only if both RNA templates were used for the generation of minus strand DNA. In contrast, only a single minus strand DNA appeared to be used as template for the plus strand DNA in the generation of fully double-stranded viral DNA. These results indicate that the first strand transfer step in reverse transcription is an intermolecular event while that of the second transfer is intramolecular. Thus, retroviruses contain two functionally active RNA's, and both may be required for the generation of a single linear DNA molecule. Formation of heterozygotes during retrovirus infection would be expected to result in the efficient generation of LTR recombinants.
217 citations
TL;DR: The nucleotide sequence of the rpoB gene of Salmonella typhimurium was compared with known sequences from other sources and the conservative regions were detected, allowing some interesting conclusions to be made about the distribution of the functional domains in bacterial RNA polymerase and about the three-dimensional structure of its β subunit.
Abstract: The nucleotide sequence of the rpoB gene of Salmonella typhimurium has been determined in this work. It was compared with known sequences of the gene from other sources and the conservative regions were detected. This allowed some interesting conclusions to be made about the distribution of the functional domains in bacterial RNA polymerase and about the three-dimensional structure of its β subunit.
TL;DR: It is concluded that the carboxyl-terminal domain is essential for RNA polymerase II activity, since substantial mutations in this region result in loss of function.
Abstract: The carboxyl-terminal domain (CTD) of the mouse RNA polymerase II largest subunit consists of 52 repeats of a seven-amino-acid block with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. A genetic approach was used to determine whether the CTD plays an essential role in RNA polymerase function. Deletion, insertion, and substitution mutations were created in the repetitive region of an alpha-amanitin-resistant largest-subunit gene. The effects of these mutations on RNA polymerase II activity were assayed by measuring the ability of mutant genes to confer alpha-amanitin resistance after transfection of susceptible rodent cells. Mutations that resulted in CTDs containing between 36 and 78 repeats had no effect on the transfer of alpha-amanitin resistance, whereas mutations with 25 or fewer repeats were inactive in this assay. Mutations that contained 29, 31, or 32 repeats had an intermediate effect; the number of alpha-amanitin-resistant colonies was lower and the colonies obtained were smaller, indicating that the mutant RNA polymerase II was defective. In addition, not all of the heptameric repeats were functionally equivalent in that repeats that diverged in up to three amino acids from the consensus sequence could not substitute for the conserved heptamer repeats. We concluded that the CTD is essential for RNA polymerase II activity, since substantial mutations in this region result in loss of function.
TL;DR: The ability to generate large amounts of cDNA from platelet-specific mRNA sequences should make possible direct molecular characterization of normal platelet proteins, and facilitate the investigation of a wide variety of inherited platelet disorders.
Abstract: Human platelets are derived from megakaryocytes as anucleate cells, and thus contain only vestigial amounts of RNA capable of being transcribed into protein. This has greatly hampered efforts to study directly platelet-specific gene products and their associated polymorphisms. In this report, we describe direct amplification, using the polymerase chain reaction, of platelet-derived mRNA in amounts sufficient to permit detailed analysis, such as restriction mapping and nucleotide sequencing. The ability to generate large amounts of cDNA from platelet-specific mRNA sequences should make possible direct molecular characterization of normal platelet proteins, and facilitate the investigation of a wide variety of inherited platelet disorders.
TL;DR: Experiments with the WSN influenza virus temperature-sensitive mutant ts56 containing a defect in the nucleocapsid protein proved that the nucleoprotein was indeed required for template RNA synthesis both in vivo and in vitro.
Abstract: The two steps in influenza virus RNA replication are (i) the synthesis of template RNAs, i.e., full-length copies of the virion RNAs, and (ii) the copying of these template RNAs into new virion RNAs. We prepared nuclear extracts from infected HeLa cells that catalyzed both template RNA and virion RNA synthesis in vitro in the absence of an added primer. Antibody depletion experiments implicated nucleocapsid protein molecules not associated with nucleocapsids in template RNA synthesis for antitermination at the polyadenylation site used during viral mRNA synthesis. Experiments with the WSN influenza virus temperature-sensitive mutant ts56 containing a defect in the nucleocapsid protein proved that the nucleocapsid protein was indeed required for template RNA synthesis both in vivo and in vitro. Nuclear extracts prepared from mutant virus-infected cells synthesized template RNA at the permissive temperature but not at the nonpermissive temperature, whereas the synthesis of mRNA-size transcripts was not decreased at the nonpermissive temperature. Antibody depletion experiments showed that nucleocapsid protein molecules not associated with nucleocapsids were also required for the copying of template RNA into virion RNA. In contrast to the situation with the synthesis of transcripts complementary to virion RNA, no discrete termination product(s) were made during virion RNA synthesis in vitro in the absence of nucleocapsid protein molecules.
TL;DR: The low activities resulting from substitutions and a small deletion in arm C suggest this region of the RNA to be a major feature involved in replicase binding, and nucleotides within the loop of arm C appear to be involved in a sequence-specific interaction with the replic enzyme.
TL;DR: It is reported that a Northwestern blotting protocol using radiolabeled viral RNAs in the presence of host cell competitor RNA can be used to demonstrate a high-affinity interaction between the MHV N protein and the virus-specific RNAs and suggests that this interaction may play an important role in the discontinuous nonprocessive RNA transcriptional process unique to coronaviruses.
Abstract: Northwestern blot analysis in the presence of competitor RNA was used to examine the interaction between the mouse hepatitis virus (MHV) nucleocapsid protein (N) and virus-specific RNAs. Our accompanying article demonstrates that anti-N monoclonal antibodies immunoprecipitated all seven MHV-specific RNAs as well as the small leader-containing RNAs from infected cells. In this article we report that a Northwestern blotting protocol using radiolabeled viral RNAs in the presence of host cell competitor RNA can be used to demonstrate a high-affinity interaction between the MHV N protein and the virus-specific RNAs. Further, RNA probes prepared by in vitro transcription were used to define the sequences that participate in such high-affinity binding. A specific interaction occurs between the N protein and sequences contained with the leader RNA which is conserved at the 5' end of all MHV RNAs. We have further defined the binding sites to the area of nucleotides 56 to 65 at the 3' end of the leader RNA and suggest that this interaction may play an important role in the discontinuous nonprocessive RNA transcriptional process unique to coronaviruses.
TL;DR: The results imply that these three poliovirus mutants map in a region (region P) primarily involved in viral protein synthesis and that their inability to shut off host cell translation is secondary to a quantitative defect in protein 2A.
Abstract: Twenty-one mutations were engineered in the 5' noncoding region of poliovirus type 1 RNA, using an infectious cDNA copy of the viral genome. RNA was made from these constructs and used to transfect HeLa cells. Viable virus was recovered from 12 of these transfection experiments, including six strains with a recognizable phenotype, mapping in four different regions. One mutant of each site was studied in more detail. Mutant 5NC-11, having a 4-base insertion at nucleotide 70, was dramatically deficient in RNA synthesis, suggesting that the far 5' end of the genome is primarily involved in one or more steps of RNA replication. Mutants 5NC-13, 5NC-114, and 5NC-116, mapping at nucleotides 224, 270, and 392, respectively, showed a similar behavior; they made very little viral protein, they did not inhibit host cell translation, and they synthesized a significant amount of viral RNA, although with some delay compared with wild type. These three mutants were efficiently complemented by all other poliovirus mutants tested, except those with lesions in protein 2A. Our results imply that these three mutants map in a region (region P) primarily involved in viral protein synthesis and that their inability to shut off host cell translation is secondary to a quantitative defect in protein 2A. The exact function of region P is still to be determined, but our data supports the hypothesis of a single functional module allowing viral protein synthesis and extending over several hundred nucleotides.
TL;DR: A strong correlation was found between the ability to overcome the resistance and a decrease in local net charge, suggesting the involvement of an electrostatic interaction between the viral 130 and 180 kd proteins and a putative host resistance factor.
Abstract: A resistance-breaking strain of tobacco mosaic virus (TMV), Ltal, is able to multiply in tomatoes with the Tm-1 gene, unlike its parent strain, L. Comparison of the genomic sequences of L and Lta1 revealed two base substitutions resulting in amino acid changes in the 130 and 180 kd proteins: Gln-979 --> Glu and His-984 --> Tyr. To clarify their involvement in the resistance-breaking property of Lta1, the two substitions were introduced into L by an in vitro transcription system to generate a mutant strain, T1. T1 multiplied in Tm-1/Tm-1 tomatoes with symptoms as did Lta1. Two additional mutant strains were constructed, each of which had one base substitution which caused a His-984 --> Tyr change (T2) or a Gln-979 --> Glu change (T3). T3 multiplied in tomato plants and protoplasts with the Tm-1 gene, indicating that the single base substitution is sufficient to overcome the resistance. T2 also multiplied, but its multiplication was greatly decreased. Although no sequence changes were detected in any progeny viruses recovered from plants without the Tm-1 gene, progeny viruses recovered from T2- or T3- inoculated Tm-1/Tm-1 tomatoes contained in most cases viruses with additional second base substitutions. They caused amino acid changes near the mutagenized residues, suggesting that the ability of T3 to overcome the resistance is not the same as that of Lta1. Sequencing of the genomic RNAs of other independently isolated resistance-breaking strains revealed the same two base substitutions found in the Lta1 RNA. These observations suggest that the two concomitant base substitutions, and possibly also the resulting amino acid changes, guarantee successful replication of these TMV strains in tomatoes containing the Tm-1 gene. A strong correlation was found between the ability to overcome the resistance and a decrease in local net charge, suggesting the involvement of an electrostatic interaction between the viral 130 and 180 kd proteins and a putative host resistance factor.
TL;DR: By mixing the protein and RNA fractions, primer-dependent RNA-synthesizing activity was regained and RNP (RNA polymerase-RNA-NP) complexes were reconstituted; they synthesized template-sized transcripts as did native RNP cores.
Abstract: Ribonucleoprotein (RNP) cores of influenza virus A/PR/8/34 were dissociated into RNA polymerase (PB1-PB2-PA complex)-associated genome RNA and nuclear protein (NP) fractions by CsCl centrifugation. The RNA polymerase-RNA complexes were capable of catalyzing the endonucleolytic cleavage of capped RNA, the initiation of primer-dependent RNA synthesis, and the synthesis of small-sized RNA, but were unable to synthesize template-sized RNA. By adding the NP protein to the RNA polymerase-RNA complexes, RNP (RNA polymerase-RNA-NP) complexes were reconstituted; they synthesized template-sized transcripts as did native RNP cores. These observations are consistent with the model where viral RNA polymerase is composed of the three P proteins while NP is essential for the elongation of RNA chains. RNP was completely dissociated into RNA-free proteins (PB1, PB2, PA, and NP) and a protein-free genome RNA fraction by centrifugation in cesium trifluoroacetate (CsTFA) and glycerol. By mixing the protein and RNA fractions, primer-dependent RNA-synthesizing activity was regained. These complexes, however, produced only small-sized RNA, presumably due to incorrect assembly of NP on viral RNA.
TL;DR: It is proposed that NS may be responsible for conferring the sequence specificity of the RNA binding that occurs during VSV genome replication.
Abstract: The interactions between the nucleocapsid protein N and either RNA or the phosphoprotein NS of vesicular stomatitis virus (VSV) were studied by the transcription of N and NS mRNAs from SP6 vectors, followed by translation in a rabbit reticulocyte lysate. Nascent N protein bound tightly to added labeled RNA, as well as to endogenous RNA in the reticulocyte lysate. This binding was demonstrated by three independent techniques. First, labeled N protein and labeled RNA migrated identically as a series of sharp, closely spaced bands in a nondenaturing gel system. Second, translated N protein behaved as a stable ribonucleoprotein complex in CsCl gradients and sedimented to the same density as the authentic N-RNA template of VSV. Third, translated N protein protected a series of labeled RNA fragments from digestion by RNase A. None of the three RNA-binding criteria was satisfied by either translated NS protein or two deletion mutants of N protein or by other components of the reticulocyte lysate. The evidence suggests that the observed binding of RNA by nascent N was not RNA sequence specific, in contrast to the encapsidation process during VSV replication. Moreover, the prior formation of N-NS complexes totally abolished the observed binding of RNA by N. Thus, we propose that NS may be responsible for conferring the sequence specificity of the RNA binding that occurs during VSV genome replication.
TL;DR: It is suggested that RNA polymerase III itself may recognize the site of transcription initiation and thus be directly involved in efficient promoter selection.
TL;DR: The fidelity of RNA replication by the poliovirus-RNA-dependent RNA polymerase was examined by copying homopolymeric RNA templates in vitro and there were no significant differences among the error frequencies obtained with different noncomplementary nucleotide substrates on a given template.
Abstract: The fidelity of RNA replication by the poliovirus-RNA-dependent RNA polymerase was examined by copying homopolymeric RNA templates in vitro. The poliovirus RNA polymerase was extensively purified and used to copy poly(A), poly(C), or poly(I) templates with equimolar concentrations of noncomplementary and complementary ribonucleotides. The error frequency was expressed as the amount of a noncomplementary nucleotide incorporated divided by the total amount of complementary and noncomplementary nucleotide incorporated. The polymerase error frequencies were very high and ranged from 7 x 10(-4) to 5.4 x 10(-3), depending on the specific reaction conditions. There were no significant differences among the error frequencies obtained with different noncomplementary nucleotide substrates on a given template or between the values determined on two different templates for a specific noncomplementary substrate. The activity of the polymerase on poly(U) and poly(G) was too low to measure error frequencies on these templates. A fivefold increase in the error frequency was observed when the reaction conditions were changed from 3.0 mM Mg2+ (pH 7.0) to 7.0 mM Mg2+ (pH 8.0). This increase in the error frequency correlates with an eightfold increase in the elongation rate that was observed under the same conditions in a previous study.
TL;DR: Analysis by immunoprecipitation of viral proteins induced after transfection of R1 RNA into HeLa cells revealed the presence of proteins 2Apro, 2C, and 3Dpol and its precursors, suggesting that the polyprotein cleavages are similar to those occurring in virus-infected cells.
Abstract: Poliovirus RNAs containing in-frame deletions within the capsid-coding region were produced by in vitro transcription of altered poliovirus type 1 cDNA by using bacteriophage T7 RNA polymerase. Three RNAs were transcribed that contained deletions of 2,317 nucleotides (bases 747 to 3064), 1,781 nucleotides (bases 1,175 to 2,956), and 1,295 nucleotides (bases 1,175 to 2,470). All three subgenomic RNAs replicated after transfection into HeLa cells, demonstrating that sequences encoding the capsid polypeptides are not essential for viral RNA replication in vivo. Viral RNA containing the largest deletion (R1) replicated approximately three times better than full-length RNA produced in vitro. Northern blot (RNA blot) hybridization analysis of total cellular RNA from HeLa cells at different times after transfection with R1 demonstrated the presence of increasing amounts of the expected 5.1-kilobase subgenomic RNA. Analysis by immunoprecipitation of viral proteins induced after transfection of R1 RNA into HeLa cells revealed the presence of proteins 2Apro, 2C, and 3Dpol and its precursors, suggesting that the polyprotein cleavages are similar to those occurring in virus-infected cells. Replication of P2/Lansing virion RNA was inhibited by cotransfection with the R1 replicon, as demonstrated by hybridization analysis with a serotype-specific oligonucleotide probe. A higher level of inhibition of RNA replication was observed when P2/Lansing RNA was cotransfected into HeLa cells with truncated R1 transcripts (R1-PvuII) that were missing 395 3' nucleotides and a poly(A) tail. These internally and terminally deleted RNAs inhibited the replication of subgenomic replicons R1, R2, and R3 and caused a reduction in plaque size when cotransfected with P1/Mahoney or P2/Lansing viral RNA, suggesting that individual cells had received both RNAs. No inhibition of plaque size was observed when replicon RNAs were used that were missing 1,384 or 1,839 3' nucleotides or contained plasmid-derived sequences downstream of the 3' poly(A). The trans-acting inhibitory effect of R1-PvuII on the replication of poliovirus P2/Lansing RNA did not involve entry of RNA into cells and appeared to reduce viral translation and RNA synthesis late in the infection cycle.
TL;DR: It is concluded that most RNAs are degraded exonucleolytically from the 3' end, but specialized internal sequences can greatly destabilize the RNA, possibly by acting as a site for an endonuclease.
Abstract: Synthetic capped RNA transcripts injected into fertilized eggs of Xenopus laevis have a half-life of 3-4 h. Addition of a long (approximately 200 nucleotide) poly(A) tail increases the half-life to 6-8 h which approaches the half-life of natural polyadenylated globin RNA injected into embryos. Since exonucleolytic action alone could account for the degradation of RNA, we tested whether circular RNA is stable after injection and find that circles are exceptionally stable (half-life greater than 40 h). After the midblastula transition, polyadenylated chloramphenicol transferase (CAT) mRNAs transcribed from injected plasmids have a half-life of 2.5 h. Insertion of a 1000 nucleotide 3' untranslated region from the Xhox-36 gene into the transcripts does not affect the half-life. In contrast to the finding that internal sequences do not affect stability, we find that sequences from the TFIIIA message reduce the half-life of CAT mRNA from 2.5 h to less than 30 min. We conclude that most RNAs are degraded exonucleolytically from the 3' end, but specialized internal sequences can greatly destabilize the RNA, possibly by acting as a site for an endonuclease.
TL;DR: Homologies to sequences surrounding the initiation sites of subgenomic RNAs from several plant RNA viruses, and from alphaviruses, have been detected.
Abstract: The subgenomic promoter of a (+)-stranded RNA virus, brome mosaic virus (BMV) controlling synthesis of subgenomic RNA4 has been defined in vitro. Truncated and mutant (-)-strand RNA templates were produced by in vitro transcription of cloned RNA3 cDNA. Subgenomic (+)-sense RNA was synthesized in vitro from these templates by a replicase (RNA-dependent RNA polymerase) preparation extracted from infected barley leaves. The activities of templates with truncations and deletions surrounding the RNA4 initiation site revealed a promoter of approximately 62 bases grouped into four functional domains. The core sequence consists of about twenty bases immediately upstream of, and including, the initiation nucleotide. In addition to the core sequence, a domain overlapping the 5' untranslated end of RNA4 apparently determines correct initiation. Two domains immediately upstream of the promoter core consist of the internal poly(A) tract of RNA3, which probably serves as an non base-paired spacer facilitating access of the replicase to the promoter, and a sequence, UUAUUAUU, that is required for high levels of promoter activity. Homologies to sequences surrounding the initiation sites of subgenomic RNAs from several plant RNA viruses, and from alphaviruses, have been detected.
TL;DR: Indirect immunofluorescence shows that purified T7 RNA polymerase, when microinjected into monkey kidney (Vero) cells, localizes predominantly in the cytoplasm while fusion proteins having only foreign codons ahead of codon 11 also have transcription activity in E. coli.
TL;DR: It is shown here that the activator RNA has the properties of dsRNA: it chromatographs with dsRNAs, can be denatured and reannealed, and is destroyed by a ds RNA-specific nuclease.
TL;DR: Under the conditions measured, the rate of association of RNA polymerase with rrnB P1 is as rapid as, or more rapid than, that reported for any other E. coli or bacteriophage promoter.
Abstract: We have established conditions that stabilize the interaction between RNA polymerase and the rrnB P1 promoter in vitro. The requirements for quantitative complex formation are unusual for E. coli promoters: (1) The inclusion of a competitor is required to allow visualization of a specific footprint. (2) Low salt concentrations are necessary since complex formation is salt sensitive. (3) The addition of the initiating nucleotides ATP and CTP, resulting in a low rate of dinucleotide production, is required in order to prevent dissociation of the complexes. The complex has been examined using DNAase I footprinting and filter binding assays. It is characterized by a region protected from DNAase I cleavage that extends slightly upstream of the region protected by RNA polymerase in most E. coli promoters. We find that only one mole of active RNA polymerase is required per mole of promoter DNA in order to detect filter-bound complexes. Under the conditions measured, the rate of association of RNA polymerase with rrnB P1 is as rapid as, or more rapid than, that reported for any other E. coli or bacteriophage promoter.