Showing papers on "RNA-dependent RNA polymerase published in 1976"
TL;DR: The complete, primary chemical structure of a viral genome has now been established and biological properties, such as ribosome binding and codon interactions can now be discussed on a molecular basis.
Abstract: Bacteriophage MS2 RNA is 3,569 nucleotides long. The nucleotide sequence has been established for the third and last gene, which codes for the replicase protein. A secondary structure model has also been proposed. Biological properties, such as ribosome binding and codon interactions can now be discussed on a molecular basis. As the sequences for the other regions of this RNA have been published already, the complete, primary chemical structure of a viral genome has now been established.
698 citations
TL;DR: The DNAase digestion end-product of calf thymus DNA contains oligonucleotides that will function as primers for the efficient transcription into DNA of many naturally-occurring RNA's by purified avian sarcoma virus RNA-directed DNA polymerase.
643 citations
TL;DR: The only potential 5'-terminal m7G is pUp as discussed by the authors, which is present in a yield of 1 mole/mole of poliovirus RNA and is not required for translation of at least one RNA species in animal cells.
Abstract: Poliovirus RNA purified from virus-specific polyribosomes does not contain m7G in a 5'-5'-pyrophosphate linkage at its 5'-end The only potential 5'-end found in ribonuclease digests of this RNA is pUp, which is present in a yield of 1 mole/mole of poliovirus RNA We conclude that a 5'-terminal m7G is not required for translation of at least one RNA species in animal cells
223 citations
TL;DR: The structural polypeptides of fowl plague virus (influenza A) and those synthesized in fowl Plague virus-infected chick embryo fibroblasts have been analyzed by high resolution polyacrylamide gel electrophoresis and shown to be distinct by tryptic peptide mapping.
209 citations
TL;DR: A virus-coded low molecular weight RNA (5.2S), which migrates slightly faster on polyacrylamide gels than the well characterized adenovirus-specific 5.5S RNA, has been isolated from cells infected with adenOVirus type 2.
194 citations
TL;DR: The genomic RNA of the avian influenza A virus, fowl plague, was fractionated into eight species by electrophoresis in polyacrylamide-agarose gels containing 6 M urea and it was demonstrated that each species has a distinct nucleotide sequence.
Abstract: The genomic RNA of the avian influenza A virus, fowl plague, was fractionated into eight species by electrophoresis in polyacrylamide-agarose gels containing 6 M urea. The separated 32P-labeled RNA species were characterized by digestion with RNase T1 and fractionation of the resulting oligonucleotides by two-dimensional gel electrophoresis; this demonstrated that each species has a distinct nucleotide sequence. A tentative correlation of each genome RNA species with the virus protein that it encodes was made.
191 citations
160 citations
TL;DR: Results indicate that this small RNA molecule probably represents the intitiated lead-in RNA segment which is removed during formation of VSV mRNAs by a possible processing mechanism.
143 citations
TL;DR: This work has sequenced five RNA polymerase-protected DNA fragments that have been sequenced to date and identified those bases that make the critical contacts responsible for maintaining the polymer enzyme-DNA interaction.
Abstract: INTRODUCTION The RNA polymerase transcribes DNA into RNA starting at sites called promoters. At these sites, the polymerase must recognize a DNA sequence, attach, open the DNA strands to gain access to the bases, and then initiate transcription. How can we identify the features that the polymerase detects? One approach, pioneered by Heinz Schaller and co-workers (Schaller, Gray and Herrmann 1975), is to isolate and sequence a DNA fragment protected by the RNA polymerase against digestion with pancreatic DNase. The very stable complexes of RNA polymerase and DNA that form at 37°C (and are even maintained in DNase) define a limited number of “binding sites” corresponding to initiation sites. The sequence of the protected DNA fragments from these complexes presumably contains those bases that make the critical contacts responsible for maintaining the polymerase-DNA interaction. A more basic approach, however, is to exploit the original genetic definition of a promoter, i.e., the locus of mutations that block or enhance transcription. Changes in mutant sequences should identify the essential base pairs. PROMOTER SEQUENCES Polymerase-protected Fragments from Promoters Figure 1 shows the five RNA polymerase-protected fragments that have been sequenced to date. These protected fragments have common properties. They are about the same size, 41–44 base pairs long. In each case, the polymerase can initiate on the DNA contained in the DNase-resistant complex and synthesize a short RNA as it runs off the end of the DNA fragment. These RNAs are about 17–20 bases long and correspond to the 5′ initial...
141 citations
TL;DR: Results suggest that although the nucleocapsids appear as closed loops, the viral RNA has free 5' and 3' ends and is, therefore, not circular.
Abstract: La Crosse (LAC) virions purified by velocity and equilibrium gradient centrifugation contained three single-stranded RNA species The three segments had sedimentation coefficients of 31S, 25S, and 12S by sodium dodecyl sulfate-sucrose gradient centrifugation By comparison with other viral and cellular RNA species, the LAC viral RNAs had molecular weights of 29 x 10(6), 18 x 10(6), and 04 x 10(6) Phenol-sodium dodecyl sulfate-extracted LAC virion RNA was not infectious for BHK-21 cell cultures under conditions in which Sindbis viral RNA was infectious Treatment of LAC virus with the nonionic detergent Triton X-100 and salt released three nucleocapsid structures, each containing one species of virion RNA The nucleocapsids had sedimenation coefficients of 115S, 90S, and 65S Negative-contrast electron microscopy of the nucleocapsids indicated that they were convoluted, supercoiled, and apparently circular They had a mean diameter of 10 to 12 nm and modal lengths of 200, 510, and 700 nm (some were even longer) By chemical and enzymatic analysis of purified viral RNA, one type of 5' nucleotide (pppAp) present in the proportion of one per RNA segment was identified After periodate oxidation, each virion RNA species was labeled by reduction with [3H]sodium borohydride Taken together, these results suggest that although the nucleocapsids appear as closed loops, the viral RNA has free 5' and 3' ends and is, therefore, not circular
136 citations
TL;DR: The results suggest that HF and possibly S1, through their interaction with the 3'-terminal region of Q beta RNA, are directly involved in the recognition of the 6' end of Qbeta RNA by Qbeta replicase.
TL;DR: Oligonucleotide fingerprinting on slabs of polyacrylamide gel has been used to investigate the sequence relationships between the 42 S RNA genome of standard Semliki Forest virus and the intracellular virus-specified single-stranded RNAs isolated from baby hampster kidney cells infected withstandard virus and co-infected with standard virus and defective-interfering particles.
TL;DR: The synthesis of large, possibly complete, complementary DNA (cDNA) copies of poliovirus RNA by avian myeloblastosis virus DNA polymerase is described.
Abstract: The synthesis of large, possibly complete, complementary DNA (cDNA) copies of poliovirus RNA by avian myeloblastosis virus DNA polymerase is described. The cDNA consists of two size classes, the larger of which is approximately 7500 nucleotides. In the presence of excess deoxynucleoside triphosphates, ribonucleoside triphosphates, or sodium pyrophosphate, only the larger material is obtained. Yields of the large cDNA are 50-75% of the input RNA.
TL;DR: Host protein synthesis in poliovirus-infected HeLa cells is interrupted, but the host mRNA appears to remain completely intact and unmodified, and the difference in the 5' ends of the host cell and viral RNA could be the basis of selective translation of viral RNA during infection.
Abstract: Host protein synthesis in poliovirus-infected HeLa cells is interrupted, but the host mRNA appears to remain completely intact and unmodified. The average size and poly (A) content of host mRNA was previously known to be unchanged (Koschel, 1974; Leibowitz and Penman, 1971), and this was confirmed. In addition, the 5' terminal methylated "cap" structures remained intact, and no further base modifications at the level of 1 base in 1,000 could be detected. Poliovirus RNA from viruses was previously shown not to have "caps" (Wimmer, 1972), and in this work poliovirus RNA from polyribosomes was found to have pUp at its 5' end. Since, initiation of protein synthesis is probably the basis for the inhibition of cellular protein synthesis in infected cells, the difference in the 5' ends of the host cell and viral RNA could be the basis of selective translation of viral RNA during infection.
TL;DR: The concentrations, in copies per cell, of viral RNA sequences complementary to different regions of the genome were determined at 8, 18 and 32 hours after infection of human cells with adenovirus type 2 to account for the inability to detect, by filter hybridization, certain classes of early mRNA sequences during the late stage of infection.
TL;DR: ppGpp alters the initiation specificity of RNA polymerase holoenzyme in vitro in a direction which mimics the stringent response in vivo, and implies thatRNA polymerase can discriminate between different types of promoter.
Abstract: ppGpp alters the initiation specificity of RNA polymerase holoenzyme in vitro in a direction which mimics the stringent response in vivo. The transition temperature for opening rRNA promoters is increased by the nucleotide, that for opening ϕ80 promoters is unaffected. This implies that RNA polymerase can discriminate between different types of promoter. ppGpp may act by effecting a structural change in the enzyme.
TL;DR: RNA polymerase is an allosteric enzyme whose activity and specificity are controlled by interaction with a nucleotide, tRNA and proteins and the availability of these effectors should ensure that the quality of transcription is tightly coupled both to translation and to the metabolic state of the cell.
Abstract: RNA polymerase is an allosteric enzyme whose activity and specificity are controlled by interaction with a nucleotide, tRNA and proteins In E coli the availability of these effectors should ensure that the quality of transcription is tightly coupled both to translation and to the metabolic state of the cell
TL;DR: The ability to make long, representative DNA transcripts of tumor virus RNA provides a source of excellent probes for molecular hybridization.
Abstract: Virions of Moloney murine leukemia virus can synthesize two classes of DNA molecules complementary to their 70S RNA. One class consists of molecules about 200 nucleotides long, which are of limited sequence complexity; these molecules are formed preferentially if the dNTP concentration during the reaction is low. The second class consists of very heterogeneous DNA molecules with weight-average size of about 1,000 nucleotides containing at least 70% of the viral RNA sequences in approximately equal concentration. The longest of these molecules can be 5,000 nucleotides long. This second class of DNA is formed in large amounts only in reactions containing dNTP concentrations of 0.2 mM or higher. In such reactions after 24 h of incubation, at least 35% of the input RNA is represented in DNA copies. The ability to make long, representative DNA transcripts of tumor virus RNA provides a source of excellent probes for molecular hybridization.
TL;DR: Results indicate that preferential transcription of certain chromatin genes relative to total RNA synthesis can occur and that this process is dependent on the presence of chromosomal proteins.
TL;DR: Total reconstitution experiments performed under various conditions revealed that 5S RNA plays an important role during the last assembly step in vitro leading to an active 50S particle, demonstrating the importance of the 5SRNA in generating an active ribosomal conformation.
Abstract: Total reconstitution experiments performed under various conditions revealed that 5S RNA plays an important role during the last assembly step in vitro leading to an active 50S particle. For the preceding steps this RNA species is dispensable. However, 50S RNA can be integrated efficiently during any of the assembly steps in vitro. The 47S particle, reconstituted in two steps and lacking 5S RNA, shows low but significant activity in many functional tests. High activity could be obtained by incubating this particle with 5S RNA alone, demonstrating the importance of the 5S RNA in generating an active ribosomal conformation. In particular, the activity of the peptidyltransferase (peptidyl-tRNA:aminoacyl-tRNA N-peptidyltransferase; EC 2.3.2.12) center is drastically influenced by 5S RNA. No significant factor-dependent tRNA binding to the A-site was observed with the 47S particle, in contrast to the corresponding P-site binding. The elongation factor G dependent GTPase activity was not affected by the lack of 5S RNA.
TL;DR: Investigation of its biosynthesis and of concomitant changes in the 26 S RNA molecule show that after transcription and processing to 26 S size the large rRNA molecule is first submitted to a central cleavage in the nucleus; it is then transported to the cytoplasm where further maturation cleavages generate simultaneously 5.8 S and 2 S RNAs.
TL;DR: The proteins synthesized in vitro in response to 42S and 26S RNAs from Semliki Forest virus were labeled with formyl-[35S]methionine from initiator tRNA, and only one labeled peptide was detected after digestion with trypsin, suggesting that nonstructural proteins as large as 150,000 daltons are probably synthesized from one initiation site on the 42S RNA.
Abstract: The proteins synthesized in vitro in response to 42S and 26S RNAs from Semliki Forest virus were labeled with formyl-[35S]methionine from initiator tRNA. One protein which comigrated with viral capsid protein was labeled under the direction of 26S RNA, and only one labeled peptide was detected after digestion with trypsin. Further digestion with pronase gave rise to the dipeptide fMet-AsN. Several labeled polypeptides were found in the 42S RNA directed product and these had molecular weights of up to 150,000. However, tryptic digestion of the product yielded only one formylmethionyl-labeled peptide, which had a different mobility from that directed by the 26S RNA. Further digestion with pronase gave a single dipeptide, fMet-Ala. This indicates that nonstructural proteins as large as 150,000 daltons are probably synthesized from one initiation site on the 42S RNA. Translation starting from the internal initiation site on the 42S RNA, which is equivalent to that on the 26S RNA, could not be detected under the conditions used. Internal initiation sites which are similarly inactive have also been detected in other viral RNAs (e.g., brome mosaic virus, tobacco mosaic virus, and polyoma 19S RNA) and this suggests that, although eukaryotic mRNAs can contain more than one initiation site for protein synthesis, only the site nearer the 5' terminus is active in vitro.
TL;DR: RNA labeled with [methyl-3H] methionine and [14C]uridine was isolated from the cytoplasm of adenovirus-infected cells and purified by poly(U)-Sepharose chromatography and hybridization to filters containing immobilized adeovirus DNA suggested that the major mRNA species were methylated.
Abstract: RNA labeled with [methyl-3H] methionine and [14C]uridine was isolated from the cytoplasm of adenovirus-infected cells and purified by poly(U)-Sepharose chromatography and hybridization to filters containing immobilized adeovirus DNA. Analysis by dimethyl sulfoxide-sucrose gradient sedimentation suggested that the major mRNA species were methylated. 7-Methylguanosine was identified at the 5'-terminus of the advenovirus-specific RNA and could be removed by periodate oxidation and beta-elimination. Structures of the type m7G(5')ppp(5')Nm containing the unusual nucleoside N6, O2'-dimethyladenosine, and smaller amounts of 2'-O-methyladenosine were isolated by DEAE-cellulose chromatography after P1 nuclease digestion of the RNA. Evidence for some 5'-terminal sequences, m7G(5')ppp(5')m6AmpNm, with additional 2'-O-methylribonucleosides was also obtained. A base-methylated nucleoside, N6-methyladenosine, is located within the RNA chain and is released as a mononucleotide by alkali hydrolysis.
TL;DR: Both the specificity and stoichiometry of the p12-RNA interactions suggest that these RNA tumor virus proteins have a regulatory role in cells.
TL;DR: Of the various intermediate fractions in the purfication procedure, only the relatively crude post-nuclear supernatant fraction was competent to synthesize the major single-stranded RNAs found in infected cells.
Abstract: A purification method for Semliki Forest virus-specified RNA-dependent RNA polymerase from BHK cells is described. The procedure entails (i) the preparation of a crude cell lysate by Dounce homogenization of cells 3-5 h post-infection, (ii) differential centrifugation to give a 15 000 g 'mitochondrial' pellet, (iii) equilibrium centrifugation on discontinuous sucrose gradients (Friedman et al. 1972) to give a membranous band of density 1-16 g/ml, (iv) solubilization with Triton N-101 and velocity centrifugation to give a 25S solubilized polymerase complex and (v) affinity chromatography through an oligo (dT)-cellulose matrix bearing immobilized 42S virus particle RNA. The overall purification was approx. 360-fold with a 5% recovery of activity. Of the various intermediate fractions in the purfication procedure, only the relatively crude post-nuclear supernatant fraction was competent to synthesize the major single-stranded RNAs found in infected cells. Other fractions incorporated precursor only into replicative intermediate (RI) or replicative from (RF). Analysis of the product RF showed that it was of the same size and could bind to the same extent to oligo (dT)-cellulose as the RF isolated directly from lysates of infected cells. Displacement hybridization and ribonuclease digestion suggested that the purified polymerase could only complete previously initiated progeny positive strands using negative strands as template and, even in its most highly purified form, was still tightly bound to its template. Analysis on polyacrylamide slab gels revealed the presence of three 35S-labelled polypeptides in the purified polymerase preparation, but a polypeptide which had identical electrophoretic mobility to the lowest mol. wt. polypeptide of the purified polymerase was also present in material from mock-fected cells which had been taken through the purification procedure. From these results we conclude that only two virus-specified polypeptides are present in the polymerase. A scheme for the synthesis of these polypeptides is presented in the accompanying paper.
TL;DR: It is proposed that S1 functions in conjunction with initiation factor IF-3 by recognizing and unfolding elements of the tertiary structure of phage RNA.
Abstract: The previously reported requirement of ribosomal protein S1 for translation of phage RNA is now shown to be related to the involvement of the protein in initiation complex formation. The structure of the messenger RNA appears to be uniquely related to S1 function, since translation and initiation with mildly unfolded phage RNA (by modification with formaldehyde) are independent of S1. It is proposed that S1 functions in conjunction with initiation factor IF-3 by recognizing and unfolding elements of the tertiary structure of phage RNA. A model is suggested for S1 function in both initiation of protein synthesis and initiation of phage RNA replication.
TL;DR: An E. coli mutant rpoA109 unable to support the growth of phage P2 produces DNA-dependent RNA polymerase with an altered α subunit that is substituted for leucine in one tryptic peptide from the mutant α sub unit.
Abstract: An E. coli mutant rpoA109 unable to support the growth of phage P2 produces DNA-dependent RNA polymerase with an altered α subunit. Histidine is substituted for leucine in one tryptic peptide from the mutant α subunit. The existence of only one rpoA gene within the E. coli chromosome is indicated.
TL;DR: It is suggested that the first step in the genesis of sarcoma viruses involves the packaging of this expressed Sarcoma virus-specific RNA in helper viral particles and that efficient transmission of the sarcomA virus- specific RNA requires additional events.
Abstract: Various rat cell lines have been analyzed for expression of endogenous RNA homologous either to RT21C, a typical rat type C virus, or to Kirsten sarcoma virus. Cells have been found that express either (i) high levels of RNA homologous to RT21C rat type C virus and low levels of RNA homologous to Kirsten sarcoma virus (RT21Chigh,sarclow) or (ii) high levels of RNA homologous to Kirsten sarcoma virus and low levels of RNA homologous to typical rat type C virus (sarchigh, RT21Clow). The properties of these two classes of cell lines have been compared. Each type of cell contains an equal amount of the expressed RNA on polysomes. Cell lines that are RT21Chigh produce abundant rat p30 nad p12 structural proteins and release rat type C particles containing viral RNA and reverse transcriptase into supernatant fluids from these cultures. Cell lines that are sarchigh,RTC21Clow have no detectable rat viral p12 protein and no p30 protein immunoreactive in even broad interspecies radioimmunoassays, and do not release type C particles into the supernatant from the cultures. When the particle-negative cell lines are superinfected with heterologous mouse or wooly type C viruses or are producing typical rat type C virus particles, the endogenous sarcoma virus-specific RNA is secreted from these cells. The sarcoma virus-specific RNA can be transcribed in complementary DNA in the endogenous reverse transcriptase reactions carried out in vitro with such virus preparations. However, exposure of cells that are permissive to the helper virus with the particles containing sarcoma virus-specific RNA has not yet resulted in cell transformation or in the synthesis of these RNA sequences. The results suggest: (i) that the first step in the genesis of sarcoma viruses involves the packaging of this expressed sarcoma virus-specific RNA in helper viral particles; (ii) that efficient transmission of the sarcoma virus-specific RNA requires additional events; and (iii) that the formation of a stable sarcoma virus by recombination between the helper viral genome and part of the rescued sarcoma virus-specific RNA is much less common event than the rescue process itself.
TL;DR: Yeast RNA polymerase I and III show a pattern of alpha-amanitin sensitivity that is the reverse of that seen for the analogous vertebrate RNA polymerases.
Abstract: Three peaks of DNA-dependent RNA polymerase (RNA nucleotidyltransferase) activity are resolved by chromatography of a sonicated yeast cell extract on DEAE-Sephadex. The enzymes, which are named RNA polymerases I, II, and III in order of elution, show similar catalytic properties to the vertebrate class I, class II, and class III RNA polymerases, respectively. Yeast RNA polymerase III is readily distinguished from yeast polymerase I by its biphasic amnonium sulfate activation profile with native DNA templates, greater enzymatic activity with poly[d(I-C)] than with native salmon sperm DNA, and distinctive chromatographic elution positions from DEAE-cellulose (0.12 M ammonium sulfate) compared with DEAE-Sephadex (0.32 M ammonium sulfate). The three yeast RNA polymerases also show significant differences in alpha-amanitin inhibition. RNA polymerase II is the most sensitive (50% inhibition at 1.0 mug of alpha-amanitin per ml). Contrary to the results for vertebrate systems, yeast polymerase I can be completely inhibited by alpha-amanitin at high concentrations (50% inhibition at 600 mug/ml) while yeast RNA polymerase II BEGINS TO SHOW SIGNIFICANT INHIBITION ONLY AT CONCENTRATIONS EXCEEDING 1 MG/ML. Therefore, yeast RNA polymerases I and III show a pattern of alpha-amanitin sensitivity that is the reverse of that seen for the analogous vertebrate RNA polymerases.
TL;DR: DNA-dependent RNA polymerase has been found in all bacterial species where it has been sought, and its distribution, together with its sensitivity to drugs that inhibit bacterial transcription, indicates that it is the enzyme responsible for the major part of RNA synthesis in the bacterial cell.
Abstract: BACKGROUND Transcription of genetic sequences is mediated by DNA-dependent RNA polymerase. The enzyme catalyzes the initiation, elongation and termination of polyribonucleotide chains employing ribonucleoside triphosphates as substrates. The synthetic reaction shows an absolute requirement for a divalent metal ion and normally requires the presence of DNA or a polydeoxyribonucleotide to serve as a template in the reaction. Incorporation of nucleotidyl residues from ribonucleoside triphosphates into an RNA-like material was first reported in 1959 by Weiss and Gladstone in a nuclear system from rat liver (see also Weiss, this volume). The bacterial enzyme was identified in several laboratories shortly thereafter and was shown to be DNA-dependent. Bacterial RNA polymerase was subsequently purified from a number of bacterial species, including, initially, Escherichia coli, Microccus luteus and Azotobacter vinelandii. The enzyme from E. coli has been the one most extensively studied, although the RNA polymerases of other bacteria, including species of Azotobacter, Bacillus, Pseudomonas and Caulobacter, have also been well characterized. DNA-dependent RNA polymerase has been found in all bacterial species where it has been sought, and its distribution, together with its sensitivity to drugs that inhibit bacterial transcription, indicates that it is the enzyme responsible for the major part of RNA synthesis in the bacterial cell. A newly reported bacterial RNA polymerase, the dnaG gene product, which is not related to the classic enzyme, seems to be involved in initiation of DNA synthesis for certain replicons (Kornberg, this volume); however, this enzyme is not known to play a role in general transcription.