Showing papers on "RNA-dependent RNA polymerase published in 1983"
TL;DR: The identity of genes that specify the largest RNA polymerase II subunits, the 220-000- and 150,000-dalton polypeptides, was confirmed by competitive radioimmune assay.
Abstract: Genes encoding yeast RNA polymerase II subunits were cloned. Efficient isolation of these genes was accomplished by probing a phage lambda gt11 recombinant DNA expression library with polyvalent antibodies directed against purified yeast RNA polymerase II. The identity of genes that specify the largest RNA polymerase II subunits, the 220,000- and 150,000-dalton polypeptides, was confirmed by competitive radioimmune assay. Both of these genes exist in single copy in the yeast Saccharomyces cerevisiae.
952 citations
TL;DR: No strict correlation exists between enzyme sensitivity and MIC values, since inhibition of RNA synthesis does not always show up to the same extent in the two different test systems used for the determination of these values.
Abstract: Rifampin specifically inhibits bacterial RNA polymerase, the enzyme responsible for DNA transcription, by forming a stable drug-enzyme complex with a binding constant of 10(-9) M at 37 C. The corresponding mammalian enzymes are not affected by rifampin. Bacterial resistance to rifampin is caused by mutations leading to a change in the structure of the beta subunit of RNA polymerase. Such resistance is not an all-or-nothing phenomenon; rather, a large number of RNA polymerases with various degrees of sensitivity to rifampin have been found. No strict correlation exists between enzyme sensitivity and MIC values, since inhibition of RNA synthesis does not always show up to the same extent in the two different test systems used for the determination of these values.
345 citations
TL;DR: Oligonucleotide map analysis of the large (L), medium (M), and small (S) genome segments of Hantaan virus demonstrated that each RNA species was unique with respect to each other and was different from host cell ribosomal RNA.
269 citations
TL;DR: The intervening sequence of the Tetrahymena ribosomal RNA precursor is excised as a linear RNA molecule which subsequently cyclizes itself in a protein-independent reaction, analogous to that by which splicing of the precursor RNA is achieved.
Abstract: The intervening sequence (IVS) of the Tetrahymena ribosomal RNA precursor is excised as a linear RNA molecule which subsequently cyclizes itself in a protein-independent reaction. Cyclization involves cleavage of the linear IVS RNA 15 nucleotides from its 5' end and formation of a phosphodiester bond between the new 5' phosphate and the original 3'-hydroxyl terminus of the IVS. This recombination mechanism is analogous to that by which splicing of the precursor RNA is achieved. The circular molecules appear to have no direct function in RNA splicing, and we propose the cyclization serves to prevent unwanted RNA from driving the splicing reactions backwards.
257 citations
TL;DR: The nucleotide sequences around the origins of related plasmids p15A, ColE1, pBR322, RSF1030 and CloDF13 are compared, and the significance of possible RNA secondary structures in primer formation is discussed.
179 citations
TL;DR: The action of s Sigma 28-RNA polymerase appears to be regulated by the spoO genes and the functions controlled by sigma 28-promoters may be closely tied to the system involved in the initiation of sporulation.
172 citations
TL;DR: The leader RNA transcript of vesicular stomatitis virus (VSV) has been immunoprecipitated from infected BHK cell extracts by anti-La specific sera from patients with systemic lupus erythematosus (SLE).
168 citations
TL;DR: The structure of mouse hepatitis virus replicative intermediates isolated by 2 M NaCl precipitation and Sepharose 2-B column chromatography suggests that the leader RNA is utilized as a primer for mice hepatitis virus RNA transcription and is not added to mRNA post-transcriptionally.
Abstract: Mouse hepatitis virus A59 codes for seven mRNAs in infected cells. These mRNAs are transcribed from a minus (-) strand template of genome length and contain a leader RNA at their 5' ends. To further elucidate the mechanism of coronavirus transcription, we examined the structure of mouse hepatitis virus replicative intermediates (RIs) isolated by 2 M NaCl precipitation and Sepharose 2-B column chromatography. Purified RIs migrated as a single species on agarose gels and sedimented between 12 and 38S on 10 to 25% sucrose gradients. The complexes were readily heat denatured into a heterogeneous population of smaller RNA molecules which probably represent nascent plus (+) strands. RNase A digestion of RIs produced a single replicative form which sedimented between 30 and 32S. These data suggest that the RI is composed of a single genome-sized (-) strand hydrogen bonded to an average of 4 to 6.5 nascent (+) strands. In contrast, a column-purified replicative form was extremely resistant to RNase A digestion and heat denaturation and migrated as a single RNA species on agarose gels and sucrose gradients. Oligonucleotide fingerprinting of an RI revealed the presence of the 5' leader RNA on the nascent (+) strands. In addition, an average of 6.2 cap structures were present in each RI, which agrees with the average number of nascent (+) strands per RI. These data suggest that the leader RNA is utilized as a primer for mouse hepatitis virus RNA transcription and is not added to mRNA post-transcriptionally.
154 citations
TL;DR: It is concluded that the pure enzyme can recognize the simple consensus sequence found at the end of genes transcribed by RNA polymerase III, which is recognized in vivo and in complete transcription extracts.
153 citations
TL;DR: The way in which the aminoacyl acceptor arms of these tRNA‐like structures are constructed reveal novel features in RNA folding which may have a bearing on the secondary and tertiary structures of RNA in general.
Abstract: Various plant viral RNAs possess a 3' terminus with tRNA-like properties. These viral RNAs are charged with an amino acid upon incubation with the cognate aminoacyl-tRNA synthetase and ATP. We have studied the structure of end-labelled 3'-terminal fragments of turnip yellow mosaic virus RNA and brome mosaic virus RNA 2 with chemical modifications of the adenosine and cytidine residues and with enzymatic digestions using RNase T1, nuclease S1 and the double-strand-specific ribonuclease from cobra venom. The data indicate that the 3' termini of these plant viral RNAs lack a cloverleaf structure as found in classical tRNA. The three-dimensional folding, however, reveals a striking resemblance with classical tRNA. The models proposed are supported by phylogenetic data. Apparently distinct three-dimensional solutions have evolved to meet the requirements for faithful recognition by tRNA-specific enzymes. The way in which the aminoacyl acceptor arms of these tRNA-like structures are constructed reveal novel features in RNA folding which may have a bearing on the secondary and tertiary structures of RNA in general. The dynamic behaviour of brome mosaic virus RNA 2 in solution presumably is illustrative of conformational transitions, which RNAs generally undergo on changing the ionic conditions.
TL;DR: The map positions of the large RNase T1-resistant oligonucleotides of the seven mouse hepatitis virus strain A59 intracellular mRNA species were studied and provide the most direct evidence thus far for the presence of leader sequences in the mRNAs of mouse hepatitisirus, which is a cytoplasmic virus.
Abstract: To determine the structure and the mechanism of synthesis of mouse hepatitis virus mRNA, the map positions of the large RNase T1-resistant oligonucleotides of the seven mouse hepatitis virus strain A59 intracellular mRNA species were studied. We found that all but one of the oligonucleotides were mapped at the positions within each mRNA consistent with the nested-set, stairlike structure of mouse hepatitis virus mRNA (Lai et al., J. Virol. 39:823-834). However, one oligonucleotide, 10, was mapped near the 5' ends of every mRNA and virion genomic RNA. In other words, oligonucleotide 10 and, therefore, the sequences around the 5' ends of the mRNAs are not colinear with the genomic sequences. Because this oligonucleotide is present only once in the genomic RNA, this result indicates that oligonucleotide 10 is not transcribed from multiple sites on the genomic template, but rather represents a leader RNA sequence which is joined to the body sequences of the different mRNAs during mRNA transcription. This provides the most direct evidence thus far for the presence of leader sequences in the mRNAs of mouse hepatitis virus, which is a cytoplasmic virus. Several possible mechanisms of RNA synthesis are discussed.
TL;DR: It is concluded that pseudorabies virus codes for a single multifunctional immediate-early protein which is essential for the transcription of immediate- early to early RNA and is required for the continuous transcription of early (and late) RNA.
TL;DR: It is demonstrated that a heterologous RNA sequence can be copied in vitro by Q beta replicase when it is inserted into a naturally occurring Q beta replication template, using phage T4 RNA ligase.
TL;DR: The amino acid sequence deduced from the nucleotide sequence suggests that both precursor polypeptides are proteolytically cleaved at glutaminyl‐methionine and glutaminy‐glycine, respectively, to produce the two viral capsid proteins.
Abstract: Middle component RNA (M RNA) of cowpea mosaic virus (CPMV) was transcribed into cDNA and double-stranded cDNA was inserted into the EcoRI site of plasmid pBRH2. The nucleotide sequence of inserts was determined, after subcloning in bacteriophages M13mp7, M13mp8 or M13mp9, by the dideoxy chain termination method. The complete sequence of CPMV M RNA, up to the poly(A) tail, is 3481 nucleotides long. The sequence contains a long open reading frame starting at nucleotide 161 from the 5' terminus and continuing to 180 nucleotides from the 3' terminus. The sequence does not contain a polyadenylation signal for the poly(A) tail at the 3' end of CPMV RNA. The initiation site at position 161 together with AUG codons in the same reading frame at positions 512 and/or 524 account for the two large colinear precursor polypeptides translated in vitro from M RNA. The amino acid sequence deduced from the nucleotide sequence suggests that both precursor polypeptides are proteolytically cleaved at glutaminyl-methionine and glutaminyl-glycine, respectively, to produce the two viral capsid proteins.
TL;DR: Analyses of the 5' ends of snowshoe hare bunyavirus plus sense S RNA species (including mRNA) recovered from infected cells have revealed two types of termini that probably represent replicative plus sense RNA species and mRNA species that have host cell derived primer sequences.
Abstract: Analyses of the 5' ends of snowshoe hare bunyavirus plus sense S RNA species (including mRNA) recovered from infected cells have revealed two types of termini. These include ends that are essentially exact copies of the 3' end of the viral S RNA and others that are similar, but additionally have 13-14 nucleotide extensions that are heterogeneous in sequence. The former probably represent replicative plus sense RNA species, the latter mRNA species that have host cell derived primer sequences.
TL;DR: RNA extracted from the 12S "termination" factor includes an RNA species comigrating with the 60 nucleotide RNA from poly (A)- RNA, which is suggested to be present in relatively low abundance in embryos of both Psammechinus and Paracentrotus.
01 Jan 1983
TL;DR: A large body of evidence concerning changes in the surface glycoproteins, the hemagglutinin and neuraminidase, has been obtained from studies on the primary sequences, interactions with antibodies and the correlation of these data with the three-dimensional X-ray structure of these proteins.
Abstract: In this chapter the structure of the genes of influenza virus and their encoded proteins will be described. Influenza viruses contain a segmented single-stranded RNA genome which has been called “negative stranded” because the viral messenger RNAs are transcribed from the viral RNA segments. A great deal of new knowledge has been obtained about influenza A, B, and C viruses since the last major reviews of influenza virus in 1975 (Kilbourne, 1975). The complete sequence of the 8 RNA segments of influenza A virus has been obtained and this has led to a greatly increased understanding of the encoded polypeptides and how variation among influenza viruses occurs. A large body of evidence concerning changes in the surface glycoproteins, the hemagglutinin and neuraminidase, has been obtained from studies on the primary sequences, interactions with antibodies and the correlation of these data with the three-dimensional X-ray structure of these proteins.
TL;DR: The presence of multiple promoters in the hepatitis B virus genome suggests that the relative levels of viral-specific proteins detected in liver and serum may reflect differential or regulated promoter efficiency.
Abstract: We employed an in vitro cell-free transcription system to locate RNA polymerase II promoters on the hepatitis B virus genome. The strongest promoter precedes the surface antigen (HBsAg) gene, which is comprised of a long (500 base pairs) presurface region as well as the mature HBsAg coding sequence. The origin of this transcript was localized by using truncated templates and S1 endonuclease mapping. The activity of the promoter was confirmed in transfection experiments in which the complete HBsAg gene was introduced into monkey kidney cells via a simian virus 40 expression vector. A second RNA polymerase II promoter preceding the HBcAg gene was also active in the cell-free system. The presence of multiple promoters in the hepatitis B virus genome suggests that the relative levels of viral-specific proteins detected in liver and serum may reflect differential or regulated promoter efficiency.
TL;DR: It is proposed that the 115-nucleotide element of Rous sarcoma-avian leukosis virus encodes a cis-acting sequence that is necessary for the proper incorporation of viral RNA into virions.
Abstract: The v-src gene of Schmidt-Ruppin strain A of Rous sarcoma virus is flanked by a 115-nucleotide direct repeat. Mutants that lack either the upstream or downstream copy replicate normally. However, mutants that lack both copies do not replicate. Cloned viral DNA lacking both copies of the 115-nucleotide sequence is capable of directing the transcription of viral RNA posttransfection. This viral RNA is polyadenylated, spliced, exported from the nucleus, and translated into protein normally. However, virions isolated from the culture medium 48 h posttransfection lack viral RNA. When mutant DNA is contransfected with wild-type DNA, the virions produced 48 h later contain wild-type RNA but not mutant RNA, even though both RNAs are present in the cytoplasm. We propose that the 115-nucleotide element of Rous sarcoma-avian leukosis virus encodes a cis-acting sequence that is necessary for the proper incorporation of viral RNA into virions.
TL;DR: The interaction of DNA replication forks with both stationary and transcribing RNA polymerase molecules has been examined in vitro, using the multienzyme T4 bacteriophage DNA replication system and purified E. coliRNA polymerase.
TL;DR: The synthesis of 7-2 RNA in isolated nuclei exhibits a pattern of sensitivity to alpha-amanitin that is characteristic for RNA polymerase III transcription, and can be added to the list of mammalian RNase III transcripts that initially bind the La protein and then become incorporated into other types of antigenic RNP particles.
TL;DR: All of the short transcripts can be chased, with excess UTP, into 197-base run-off transcripts; thus, these RNAs are precursors of full-length transcripts and not synthetic "dead ends."
Abstract: We have synthesized RNA from cloned adenovirus 2 late promoter DNA in an in vitro transcription extract under UTP-limiting conditions. Under these circumstances, almost all of the alpha-amanitin-sensitive RNA produced is shorter than 20 nucleotides; most of these short transcripts are present in four species, 6, 7, 13, and 17 nucleotides. These short RNAs are initiated at the adenovirus 2 promoter, as judged by partial sequence analysis and by the abolition of their synthesis upon cleavage of the template DNA at sites which also abolish the production of full-length transcripts. All of the short transcripts can be chased, with excess UTP, into 197-base run-off transcripts; thus, these RNAs are precursors of full-length transcripts and not synthetic "dead ends." Significantly, none of these short RNAs is capped or 2'-O-methylated. However, 79-base run-off transcripts synthesized from this promoter with nonlimiting NTP levels are fully capped.
TL;DR: The 5' terminal sequences of the two DI RNA populations were not derived from standard Sindbis viral RNA but were almost identical to those of a cellular tRNAAsp.
Abstract: Defective interfering (DI) particles are deletion mutants that interfere specifically with the replication of homologous standard virus. We have determined the 5'-terminal nucleotide sequences of two DI RNA populations by the following methods: (i) cloning of the cDNA from one of the DI RNA populations and sequencing a representative clone, and (ii) using both DI RNA populations as templates for preparing primer-directed cDNA transcripts and sequencing these transcripts. The 5' terminal sequences of the two DI RNA populations were not derived from standard Sindbis viral RNA but were almost identical to those of a cellular tRNAAsp.
TL;DR: Five recombinant plasmids carrying rpoB rifampicin-resistant RNA-polymerase genes were obtained and sequence analysis revealed certain structural changes in the rPoB gene which specify corresponding alterations in the β-subunit of RNA polymerase.
Abstract: Five recombinant plasmids, pBK2646, pBK611, pRC3, pRC4 and pRC5, carrying rpoB rifampicin-resistant RNA-polymerase genes were obtained.
TL;DR: High level synthesis in Escherichia coli of mature human fibroblast interferon is obtained using a plasmid vector designed to allow easy coupling of a DNA coding region to the initiator AUG of the replicase gene of the RNA phage MS2 cloned downstream of phage lambda's leftward promoter.
Abstract: We have obtained high level synthesis in Escherichia coli of mature human fibroblast interferon using a plasmid vector that was designed to allow easy coupling of a DNA coding region to the initiator AUG of the replicase gene of the RNA phage MS2 cloned downstream of phage lambda's leftward promoter. The activity of the promoter can be regulated by temperature. Induced cells accumulated the interferon up to 4% of the total cellular protein. The biological activity of the product amounted to 4 X 10(9) international units per litre of culture. The synthesis of human fibroblast interferon was shown to drastically inhibit the growth rate of the bacterial host.
TL;DR: Examination of radiolabeled termini released by digestion with several base-specific RNases revealed that rotavirus RNA segments are base paired end-to-end and contain the same terminal structures.
Abstract: Both 3'- and 5'-terminal structures of human rotavirus genome double-stranded RNA segments were determined. RNAs were labeled at the 3'-termini with [32P]pCp by incubation with RNA ligase and at the 5'-termini with [32P]phosphate by polynucleotide kinase or, in the case of 5' caps, with 3H by chemical modification with [3H]NaBH4. Examination of radiolabeled termini released by digestion with several base-specific RNases revealed that rotavirus RNA segments are base paired end-to-end and contain the same terminal structures: (formula; see text)
TL;DR: The sequence of the RNA fragments shows that the nuclease causes discrete cuts in the RNA with minimal nibbling indicating that only few sites are accessible to the action of the enzyme.
Abstract: Digestion of the signal recognition particle (SRP) of dog pancreas with micrococcal nuclease results in the stepwise cleavage of the 300 nucleotide 7SL RNA moiety producing five major fragments approximately 220 (1), 150 (2), 72 (3), 62 (4) and 45 (5) nucleotides long. The RNA molecule is initially cut once yielding fragments 1 and 3. Further degradation releases fragments 2, 4 and 5. The introduction of the first nick into the 7SL RNA does not alter the structure nor the function of the SRP. Further degradation of the RNA results in disruption and loss of activity of the particle. The sequence of the RNA fragments shows that the nuclease causes discrete cuts in the RNA with minimal nibbling indicating that only few sites are accessible to the action of the enzyme. The five major products of nuclease digestion together span almost the entire length of the 7SL RNA. Nicking occurs mainly around the boundary region between the central S sequence and the flanking Alu sequences constituting the 7SL RNA (1). The S fragment is bound to the four largest polypeptides while the 5' and 3' Alu fragments are associated with the two smallest protein constituents of the SRP.
TL;DR: The sensitivity of MMTV RNA initiation and synthesis to alpha-amanitin and actinomycin D are characteristic of DNA-directed transcription by RNA polymerase II.