Showing papers on "RNA-dependent RNA polymerase published in 1975"

Nucleotide sequence of an RNA polymerase binding site at an early T7 promoter.

Abstract: Escherichia coli RNA polymerase (EC 2776), bound in a tight complex at an early T7 promoter, protects 41 to 43 base pairs of DNA from digestion by DNase I The protected DNA fragment contains both the binding site for RNA polymerase and the mRNA initiation point for the promoter The sequence of the DNA fragment and the sequence of the mRNA that it codes for are presented here A seven-base-pair sequence, apparently common to all promoters, is implicated in the formation of a tight binary complex with RNA polymerase

A new method for the large-scale purification of wheat germ DNA-dependent RNA polymerase II.

Abstract: An improved method for the purification of the alpha-amanitin-sensitive deoxyribonucleic acid dependent ribonucleic acid polymerase [ribonucleosidetriphosphate:RNA-nucleotidyltransferase, EC 2.7.7.6-A1 (RNA polymerase II or RNA polymerase B) from wheat germ is presented. The method involves homogenization of wheat germ in a buffer of moderate ionic strength, precipitation of RNA polymerase with Polymin P (a polyethylenimine), elution of RNA polymerase from the Polymin P precipitate, ammonium sulfate precipitation, and chromatography on DEAE-cellulose and phosphocellulose. RNA polymerase II is purified over 4000-fold with a 60% recovery, resulting in a yield of 25-30 mg of RNA polymerase from 1 kg of starting material.

Nucleotide sequence of an RNA polymerase binding site from the DNA of bacteriophage fd.

Abstract: The primary structure of a strong RNA polymerase binding site in the replicative form DNA of phage fd has been determined by direct DNA sequencing. It is: (see article). The molecule contains regions with 2-fold symmetry and sequence homologies to promoter regions from other DNAs. The startpoint of transcription is located in the center of the binding site.

Site on the RNA of an avian sarcoma virus at which primer is bound.

Abstract: In vitro transcription of the avian tumor virus RNA by RNA-directed DNA polymerase is initiated on the unique cellular 4S RNA. Previous studies have shown that on the average there is one such RNA primer hydrogen bonded to each viral 35S RNA. The present study confirms that finding and demonstrates that, at least for the majority of 35S RNA molecules, the primer is bound at a site close to the 5'-terminus.

Human cytomegalovirus stimulates host cell RNA synthesis.

Abstract: Human cytomegalovirus infection of human fibroblast cells (WI-38) induced cellular RNA synthesis. The RNA synthesis in infected cultures preceded the synthesis of viral DNA and progeny virus by approximately 24 h. RNA species synthesized in infected cells included ribosomal 28S and 18S; and 4S transfer RNA; all were markedly increased in comparison to uninfected cells. This induction of host cell RNA synthesis was dependent upon a protein(s) that was synthesized during the early stages of infection.

5'-Terminal m-7G(5')ppp(5')G-m-p in vivo: identification in reovirus genome RNA.

Abstract: Methylated reovirus mRNA was synthesized in vitro in the presence of S-adenosyl-L-[methyl-3H]-methionine. Viral genome double-stranded RNA that was uniformly labeled with 32-P was isolated from purified virions. The RNAs were mixed and their 5'-terminal structures compared by electrophoretic and chromatographic analyses after enzymatic digestion. Both the mRNA and the corresponding strand in the genome RNA contain m-7G(5')ppp(5')G-m-pCp, indicating that infected cells synthesize viral RNA with blocked, methylated 5' termini.

Novel initiation of RNA synthesis in vitro by vesicular stomatitis virus.

Abstract: The mRNA synthesised in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus contains the novel 5′-terminal structure G(5′)ppp(5′)Ap....

RNA synthesis in cells infected with herpes simplex virus. X. Properties of viral symmetric transcripts and of double-stranded RNA prepared from them.

Abstract: HEp-2 cells infected with herpes simplex 1 virus contain RNA transcripts capable of forming double-stranded (DS) RNA on annealing. The properties of purified DS RNA were as follows (i) DS RNA is resistant to depolymerization by RNase A or T-1 in 2 times 0.15 M NaCl, plus 0.015 M sodium citrate (SSC) but not 0.1 times SSC or following thermal denaturation. (ii) The Tm of the viral DS RNA was 100 C in 0.1 times SSC. (iii) Undenatured DS RNA does not hybridize with viral DNA: upon denaturation, excess unlabeled RNA drove 50 to 55% of labeled DNA into DNA-RNA hybrid. The kinetics of hybridization indicate that the DS RNA consists of at least two populations of transcripts arising from 29 and 26% of viral DNA and differing 40-fold in molar concentration.

Specific binding of tryptophan transfer RNA to avian myeloblastosis virus RNA-dependent DNA polymerase (reverse transcriptase)

Abstract: The ability of tryptophan tRNA (tRNATrp) to initiate reverse transcription of the 70S RNA of avian RNA tumor viruses suggested that the reverse transcriptase (RNA-dependent DNA polymerase; deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase; EC 2777) might have a specific binding site for the tRNA A complex of tRNATrp and the avian myeloblastosis virus reverse transcriptase has been demonstrated using chromatography on Sephadex G-100 columns Of all the chicken tRNAs, only tRNATrp and a tRNA4Met bind to the enzyme with high enough affinity to be selected from a mixture of the chicken cell tRNAs The ability of tRNATrp to change the sedimentation rate of the enzyme indicates that tRNATrp is not binding to a contaminant in the enzyme preparation Treatment of the enzyme with monospecific antibody to reverse transcriptase prevented binding of tRNA as well as inhibited the DNA polymerase activity of the enzyme The ability of reverse transcriptase to utilize tRNATrp aa a primer for DNA synthesis, therefore, appears to involve a highly specific site on the enzyme

Polyadenylic acid sequences in E. coli messenger RNA

Abstract: IT is commonly assumed that the messenger RNA (mRNA) of prokaryotes lacks the poly(A) sequences which characterise the 3′ terminus of most mRNA of eukaryotes. A report of the absence of poly(A) sequences from both pulse labelled and stable RNA of Escherichia coli1 is often cited as evidence for such a distinction. This apparent difference has generated speculations ranging from the role of poly(A) sequences in regulating the longevity of eukaryotic mRNA molecules1, to the evolutionary origin of mitochondria2 in which poly(A) sequences have been found in a messenger-like RNA2–5. Such speculations prompted us to re-examine evidence for AMP-rich sequences in E. coli reported6 before poly(A) sequences were known to be attached to mRNA in animal cells7–9. We have found that RNA molecules can be isolated from E. coli with the techniques developed for the isolation of poly(A) containing RNA of eukaryotes10 which have properties of mRNA and contain poly(A) sequence at their 3′ terminus.

In vivo and in vitro phosphorylation of DNA-dependent RNA polymerase of Escherichia coli by bacteriophage-T7-induced protein kinase.

Abstract: After infection with bacteriophage T7 the beta' and to a lesser extent the beta subunits of E. coli DNA-dependent RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) are phosphorylated by a phage-gene-encoded protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). The phosphorylation occurs on threonine residues and appears site-specific. It is probably the molecular basis of the early transcriptional control.

Evidence for de novo production of self-replicating and environmentally adapted RNA structures by bacteriophage Qbeta replicase.

Abstract: Highly purified coliphage Qbeta replicase when incubated without added template synthesizes self-replicating RNA species in an autocatalytic reaction. In this paper we offer strong evidence that this RNA production is directed by templates generated de novo during the lag phase. Contamination of the enzyme by traces of RNA templates was ruled out by the following experimental results: (1) Additional purification steps do not eliminate this RNA production. (2) The lag phase is lengthened to several hours by lowering substrate or enzyme concentration. At a nucleoside triphosphate concentration of 0.15 mM no RNA is produced although the template-directed RNA synthesis works normally. (3) Different enzyme concentrations lead to RNA species of completely different primary structure. (4) Addition of oligonucleotides or preincubation with only three nucleoside triphosphates affects the final RNA sequence. (5) Manipulation of conditions during the lag phase results in the production of RNA structures that are adapted to the particular incubation conditions applied (e.g., RNA resistant to nuclease attack or resistant to inhibitors or even RNAs "addicted to the drug," in the sense that they only replicate in the presence of a drug like acridine orange). RNA species obtained in different experiments under optimal incubation conditions show very similar fingerprint patterns, suggesting the operation of an instruction mechanism. A possible mechanism is discussed.

Dissociation of two polypeptide chains from yeast RNA polymerase A.

Abstract: Yeast RNA polymerase A (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) can be converted to a new form of enzyme, called RNA polymerase A*, which is lacking two polypeptide chains of 48,000 and 37,000 daltons. Apart from these two missing polypeptides the subunit structures of RNA polymerases A and A* are indistinguishable. RNA polymerase A* differs from the complete enzyme in its electrophoretic and chromatographic behavior, template requirements, and alpha-amanitin sensitivity. RNA polymerase A* transcribes the alternated copolymer d(A-T)n with the same efficiency as RNA polymerase A but its specific activity is greatly reduced with native calf thymus DNA as template. The transcription of a variety of synthetic templates is also altered by removal of the two polypeptide chains. RNA polymerase A* is inhibited by high concentrations of alpha-amanitin (500 mug/ml), whereas RNA polymerase A is comparatively less sensitive to the toxic peptide. The data are discussed in terms of possible roles of the two dissociable polypeptides.

Highly asymmetric transcription by RNA polymerase containing phage-SP01-induced polypeptides and a new host protein.

Abstract: An RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) has been purified from phage-SP01-infected Bacillus subtilis that copies RNA almost exclusively from the heavy strand of native SP01 DNA, the DNA strand from which "middle" and "late" classes of RNA are copied in vivo. Hybridization-competition established that this RNA polymerase termed enzyme A, preferentially synthesizes middle RNA in vitro. Enzyme A contains beta',beta, alpha, and two newly identified host polypeptides, variation of (21,500 daltons) and omega (11,000 daltons). All of these polypeptides are associated with highly purified RNA polymerase from uninfected bacteria. In addition, enzyme A contains phage-induced subunits of 26,000, 24,000, and 13,500 daltons. Enzyme A lacks sigma polypeptide, and strand-selective transcription by this enzyme is resistant to anti-sigma antibody. A reconstitution experiment strongly suggests that the host variation of protein is required in addition to a phage-induced subunit(s) (or an unidentified phage-induced modification) for strand-selective transcription of SP01 middle genes in vitro.

Translation of Rous sarcoma virus RNA in a cell-free system from ascites Krebs II cells.

Abstract: The template activities of the 60-70S RNA complex and of the 30-40S subunit RNA species of Rous sarcoma virus were tested in a cell-free protein-synthesizing system from mouse ascites Krebs II cells. Stimulation of protein synthesis over the endogenous background was about 2-fold with 30-40S viral RNA and about 1.3-fold with 60-70S viral RNA as template. Analysis by sodium dodecyl sulfate-gel electrophoresis showed that the predominant polypeptide synthesized in vitro in response to 30-40S RNA of Rous sarcoma virus had a molecular weight of 75,000-80,000. This polypeptide could be precipitated by antiserum against the group-specific antigens of the virus, although its molecular weight is higher than that of virion group-specific antigen proteins. Analysis of tryptic digests of the protein made in vitro indicates similarity to tryptic digests from authentic virion group-specific proteins. It is concluded that part of the RNA from Rous sarcoma virus is translated in vitro into a high-molecular-weight protein, perhaps a precursor of the virion group-specific proteins.

Adenovirus-2 mRNA is transcribed as part of a high-molecular-weight precursor RNA

Abstract: The order of transcription and the length of nascent RNA transcripts from adenovirus-2 (Ad-2) DNA in the nucleus of infected cells has been deduced by labeling the growing RNA chains in vivo for a very brief period, separating the RNA on the basis of size, and hybridizine to the ordered EcoRI restriction endonuclease fragments derived from Ad-2 DNA. The majority of the virus-specific RNA molecules are synthesized as very high-molecular-weight units beginning at a common point at least 25-30,000 base pairs from one end of the Ad-2 DNA. These molecules can be reduced in size without further RNA synthesis. The experiments indicate the obligatory origin of Ad-2 mRNA from a high-molecular-weight precursor molecule.

Influenza virion RNA-dependent RNA polymerase: stimulation by guanosine and related compounds.

Abstract: The activity of RNA-dependent RNA polymerase of several influenza viruses is stimulated by guanosine. Depending upon the virus strain used, the stimulation of initial reaction rate is up to 10-fold. 5'-GMP, 3',5'-cyclic GMP, and 5'-GDP show lesser stimulation effects. No other nucleosides of 5'-NMPs stimulate, but the dinucleoside monophosphates GpG and GpC show large stimulations. We present evidence that the stimulation represents preferential initiation of genome complementary RNA chains with guanosine: (i) [3-H] guanosine is incorporated specifically at the 5'terminus of RNA in polymerase reaction mixes in vitro. (ii) This incorporation reaction has several properties similar to those of the virion polymerase elongation reaction. (iii) RNA made in the stimulated reaction behaves as complementary RNA in annealing kinetic studies, as does RNA labeled with [3-H]guanosine.

Characteristics of the major internal protein and RNA-dependent DNA polymerase of bovine leukaemia virus.

Abstract: Summary A virus designated bovine leukaemia virus (BLV), associated with leukaemia in cattle and previously demonstrated to induce the disease in sheep, was purified from chronically infected sheep cell cultures. Electrophoretic analysis showed a major protein of mol. wt. about 24000 (p24) which reacted in gel diffusion and complement-fixation tests with sera from naturally infected cattle, experimentally infected sheep, and guinea pigs immunized with p24. BLV p24 has an isoelectric point of 8.6. Interspecies antigenic reactivities characteristic of mammalian Type C virus p30s were not detected in disrupted BLV or on p24. Sheep and guinea pig antisera to BLV, reactive withp 24, also did not precipitate several Type C virus p30s in radioimmunoassays. BLV is also distinguished from Type C viruses and resembles mouse mammary tumour virus and Mason-Pfizer virus in having an RNA-dependent DNA polymerase which is preferentially active in the presence of Mg++ when synthetic templates are used. Along with previously published morphological data, the above indicates that BLV is not a Type C virus as classically defined. Four hundred and forty one human sera from cancer patients and matched controls were non-reactive with disrupted BLV, BLV infected cells, and BLV p24 in complement-fixation tests.