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PubMed1970

Asymmetric template function of microbial deoxyribonucleic acids: transcription of ribosomal and soluble ribonucleic acids.

In Bacillus subtilis and Escherichia coli, 16 and 23S ribosomal ribonucleic acid (rRNA) hybridize exclusively with the heavy (H) strand of methylated albuminkieselguhr (MAK)-fractionated complementary deoxyribonucleic acid (DNA) strands. All the soluble RNA (4S RNA) in B. subtilis and 66 to 75% of the 4S RNA in E. coli also hybridize with the H strand. Interspecific hybridization shows that E. coli 23S rRNA also binds selectively to the DNA H strand of Salmonella typhimurium. The hybridization peak for all three cellular RNA components is specifically located in the late-eluting region of the absorbance profile of the DNA H strand. The early-eluting region of the light (L) strand preferentially inhibits the hybridization between the peak region of the H strand and 23S rRNA. These regions are considered to represent the transcribing sequences and their complements for 23S rRNA in the separated H and L strands of DNA, respectively.

Albumins↗
PubMed1971

Isoleucine auxotrophy as a consequence of a mutationally altered isoleucyl-transfer ribonucleic acid synthetase.

Among mutants which require isoleucine, but not valine, for growth, we have found two distinguishable classes. One is defective in the biosynthetic enzyme threonine deaminase (l-threonine hydro-lyase, deaminating, EC 4.2.1.16) and the other has an altered isoleucyl transfer ribonucleic acid (tRNA) synthetase [l-isoleucine: soluble RNA ligase (adenosine monophosphate), EC 6.1.1.5]. The mutation which affects ileS, the structural gene for isoleucyl-tRNA synthetase, is located between thr and pyrA at 0 min on the map of the Escherichia coli chromosome. This mutationally altered isoleucyl-tRNA synthetase has an apparent K(m) for isoleucine ( approximately 1 mm) 300-fold higher than that of the enzyme from wild type; on the other hand, the apparent V(max) is altered only slightly. When the mutationally altered ileS allele was introduced into a strain which overproduces isoleucine, the resulting strain could grow without addition of isoleucine. We conclude that the normal intracellular isoleucine level is not high enough to allow efficient charging to tRNA(Ile) by the mutant enzyme because of the K(m) defect. A consequence of the alteration in isoleucyl-tRNA synthetase was a fourfold derepression of the enzymes responsible for isoleucine biosynthesis. Thus, a functional isoleucyl-tRNA synthetase is needed for isoleucine to act as a regulator of its own biosynthesis.

Alleles↗
PubMed1971

Inhibition of arginyl-transfer ribonucleic acid synthetase activity of Escherichia coli by arginine biosynthetic precursors.

The arginine biosynthetic precursors, ornithine, citrulline, and argininosuccinate, inhibit arginyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.13, arginine: soluble RNA ligase, adenosine monophosphate) activity in the in vitro attachment assay system. Ornithine is the most potent, argininosuccinate is next, and citrulline is least effective. The implications of these results are discussed in relation to arginyl-tRNA synthetase activity and the level of the arginine biosynthetic enzymes during conditions of restricted and unrestricted supply of arginine to cells.

Amino Acyl-tRNA Synthetases↗
PubMed1971

Biochemical characterization of a mutant isoleucyl-transfer ribonucleic acid synthetase from Escherichia coli K-12.

Isoleucyl-transfer ribonucleic acid (tRNA) synthetase [l-isoleucine: soluble RNA ligase (adenosine monophosphate), EC 6.1.1.5; IRS] was partially purified from Escherichia coli K-12 and from an ileS mutant that appears to be altered in IRS. The half-life of wild-type IRS, incubated at 60.5 C, is 69 min, whereas that of mutant IRS is 8 min. Mutant IRS shows about a 100-fold lower affinity than wild-type IRS for isoleucine, dl-valine, thiaisoleucine, and O-methyl-dl-threonine, both in the pyrophosphate exchange assay and in the assay of isoleucyl-tRNA formation. The affinity of the mutant enzyme for adenosine triphosphate in the assay of isoleucyl-tRNA formation is 15-fold lower than that of the wild-type enzyme. The affinity of mutant IRS for tRNA is not changed as compared with wild-type IRS. These data show that mutant IRS has an altered structure and clearly confirm that ileS is the structural gene for IRS.

Adenosine Triphosphate↗
PubMed1973

Regulation of methionyl-transfer ribonucleic acid synthetase formation in Escherichia coli and Salmonella typhimurium.

The control of methionyl-transfer ribonucleic acid (tRNA) synthetase (l-methionine: soluble RNA ligase [adenosine monophosphate]) was studied in methionyl-tRNA synthetase mutants of Escherichia coli and Salmonella typhimurium. The results of activity determinations with crude extracts indicate that this enzyme of the E. coli mutant strain possessed a reduced affinity for methionine-tRNA, whereas this enzyme of the S. typhimurium mutant exhibited a decreased affinity for l-methionine. The differential rate of methionyl-tRNA synthetase formation in these two mutants was several-fold greater than that of the respective parental strains. On the other hand, the level of in vivo aminoacylation of methionine-tRNA was only about one-third that of the parent strains. These results suggest that aminoacylation of methionine-tRNA is a necessary step in repression control of methionyl-tRNA synthetase of both E. coli and S. typhimurium strains.

Amino Acyl-tRNA Synthetases↗
PubMed1966

Tetracycline inhibition of cell-free protein synthesis. I. Binding of tetracycline to components of the system.

Day, L. E. (Chas. Pfizer & Co., Inc., Groton, Conn.). Tetracycline inhibition of cell-free protein synthesis. I. Binding of tetracycline to components of the system. J. Bacteriol. 91:1917-1923. 1966.-Tetracycline, an inhibitor of cell-free protein synthesis, effected the dissociation of Escherichia coli 100S ribosomes to 70S particles in vivo and in vitro, but was not observed to mediate the further degradation of these particles. The antibiotic was bound by both 50S (Svedberg) and 30S subunits of 70S ribosomes and also by E. coli soluble RNA (sRNA), polyuridylic acid (poly U), and polyadenylic acid (poly A). The binding to ribosomal subunits was higher at 5 x 10(-4)m Mg(++) than at 10(-2)m Mg(++). The binding to polynucleotide chains was highest when Mg(++) was not added to the reaction mixture.

Adenine Nucleotides↗
PubMed1979

Age-related changes in function of transfer ribonucleic acid of rat livers.

Changes in transfer ribonucleic acids during aging could be caused by alterations in regulation or mutation and give rise to slower and less accurate protein synthesis. Rodent liver parenchymal cells, purified from disaggregated livers, do decrease in ability to incorporate labeled amino acids during aging. Moreover, old rodents have a rapidly degraded fraction of liver soluble RNA which is absent from middle-aged animals. In addition, tRNAs purified from old unfractionated liver cannot be acylated as well as from young. High speed supernatant tRNAs from old and young liver are quite similar in acylation capacity. Analysis indicates that a defective subfraction of tRNA may be bound to the ribosomal fraction of the liver cell. Some evidence indicates that base modification levels differ in young and old rodent liver. Shifts in the proportions of lysine and serine isoacceptors during aging are consistent with this idea. One isoacceptor change is an increase in tRNAlys4, which is correlated with cell division capacity in other systems.

Acylation↗
PubMedDate not supplied

[Metabolism of pseudouridine in rats with acute carbon tetrachloride intoxication].

After acute intoxication with CCl4 excretion of pseudouridine with urine was increased. At the some time, pseudouridine content was decreased in soluble RNA from liver tissue and unaltered in ribosomal RNA. The rate of pseudouridine turnover was studied by means of elimination of its total and specific radioactivity in urine, sRNA and rRNA. An increase in the pseudouridine excretion occurred due to acceleration in rRNA turnover and to an increase in the sRNa degradation, which was synthesized before CCL4 administration, but a decrease of pseudouridine content in sRNA was caused by inhibition of its synthesis.

Acute Disease↗
PubMed1985

Purification of a soluble template-dependent rhinovirus RNA polymerase and its dependence on a host cell protein for viral RNA synthesis.

The soluble phase of the cytoplasm of human rhinovirus type 2-infected cells contains an enzymatic activity able to copy rhinovirion RNA without an added primer. This RNA-dependent RNA polymerase (replicase) makes a specific copy of the added rhinovirion RNA, as shown by hybridization of the product to its template RNA but not to other RNAs. The same replicase preparation also contains a virus-specific polyuridylic acid [poly(U)] polymerase activity which is dependent on added polyadenylic acid-oligouridylic acid template-primer. Both activities purify together until a step at which poly(U) polymerase but no replicase activity is recovered. Addition of a purified HeLa cell protein (host factor) to this poly(U) polymerase completely reconstitutes rhinovirus replicase activity. Host factor activity can be supplied by adding oligouridylic acid, suggesting that the host cell protein acts at the initiation step of rhinovirus RNA replication. A virus-specific 64,000-dalton protein purifies with both poly(U) polymerase and replicase activities.

Cell Transformation, Viral↗
PubMed1990

Complete replication of a eukaryotic virus RNA in vitro by a purified RNA-dependent RNA polymerase.

A soluble RNA-dependent RNA polymerase was isolated from Nicotiana tabacum plants infected with cucumber mosaic virus (CMV), which has a genome of three positive-strand RNA components, 1, 2, and 3. The purified polymerase contained two virus-encoded polypeptides and one host polypeptide. Polymerase activity was completely dependent on addition of CMV RNA as template, and the products of reaction were single-stranded (ss) RNA and double-stranded (ds) RNA, corresponding to RNAs 1, 2, and 3, and a subgenomic RNA (RNA 4) derived from RNA 3. The ratio of ssRNA to dsRNA was about 5:1, and the ssRNA was shown to be predominantly the positive strand. This demonstrates the complete replication of a eukaryotic virus RNA in vitro by a template-dependent RNA polymerase.

Base Sequence↗
PubMed1982

Genome-length copies of poliovirion RNA are synthesized in vitro by the poliovirus RNA-dependent RNA polymerase.

A soluble RNA-dependent RNA polymerase was purified from the cytoplasm of poliovirus-infected HeLa cells. A single virus-specific protein designated as p63 (or NCVP4) copurified with this activity. The purified polymerase was free of ribonuclease activity and was shown to copy poliovirion RNA when oligo(U) was added to the in vitro reaction mixture. Characterization of the product RNA by electrophoresis in methylmercury (II) hydroxide-agarose gels showed that genome-sized copies of poliovirion RNA were synthesized in vitro by the purified polymerase. The product RNA was shown to be heteropolymeric, complementary to virion RNA, and covalently linked to oligo(U). The product RNA contained the expected distribution of UMP and GMP containing dinucleotide pairs which included a very low frequency of CpG pairs. The amount, size distribution, and rate of synthesis of product RNA was very dependent on the in vitro reaction conditions. Full sized product RNA was synthesized in about 6 min when reaction conditions were used that yielded maximum elongation rates (pH 8.0, 7 mM Mg2+, 37 degrees C). Under these conditions, most of the product RNA recovered from a 1-h reaction was full sized. Thus, the polymerase was found to specifically initiate synthesis at the 3'-end of the template using an oligo(U) primer and to carry out an elongation reaction at about 1250 nucleotides/min that resulted in the synthesis of full sized product RNA.

Base Sequence↗
PubMed1979

Isolation of a soluble and template-dependent poliovirus RNA polymerase that copies virion RNA in vitro.

A soluble RNA-dependent RNA polymerase was isolated from poliovirus-infected HeLa cells and was shown to copy poliovirus RNA in vitro. The enzyme was purified from a 200,000-X-g supernatant of a cytoplasmic extract of infected cells. The activity of the enzyme was measured throughout the purification by using a polyadenylic acid template and oligouridylic acid primer. The enzyme was partially purified by ammonium sulfate precipitation, glycerol gradient centrifugation, and phosphocellulose chromatography. The polymerase precipitated in a 35% saturated solution of ammonium sulfate, sedimented at about 7S on a glycerol gradient, and eluted from phosphocellulose with 0.15 M KC1. The polymerase was purified about 40-fold and was shown to be totally dependent on exogenous RNA for activity and relatively free of contaminating nuclease. The partially purified polymerase was able to use purified polio virion RNA as well as a template. Under the reaction conditions used, the polymerase required an oligouridylic acid primer and all four ribonucleside triphosphates for activity. The optimum ratio of oligouridylic acid molecules to poliovirus RNA molecules for priming activity was about 16:1. A nearest-neighbor analysis of the in vitro RNA product shows it to be heteropolymeric. Annealing the in vitro product with poliovirus RNA product shows it to be heteropolymeric. Annealing the in vitro product with poliovirus RNA rendered it resistant to RNase digestion, thus suggesting that the product RNA was complementary to the virion RNA template.

Cell-Free System↗
PubMed2006

Thermodynamics of interactions of water-soluble porphyrins with RNA duplexes.

We characterized the interactions of meso-tetrakis(4N-(2-hydroxyethyl)pyridinium-4-yl) porphyrin (TEtOHPyP4), meso-tetrakis(4N-allylpyridinium-4-yl) porphyrin (TAlPyP4), and meso-tetrakis(4N-metallylpyridinium-4-yl) porphyrin (TMetAlPyP4) with the poly(rA)poly(rU) and poly(rI)poly(rC) RNA duplexes between 18 and 45 degrees C by employing circular dichroism, light absorption, and fluorescence intensity spectroscopic measurements. Our results suggest that TEtOHPyP4 and TAlPyP4 intercalate into the poly(rA)poly(rU) and poly(rI)poly(rC) host duplexes, while TMetAlPyP4 associates with these RNA duplexes by forming outside-bound, self-stacked aggregates. We used our temperature-dependent absorption titration data to determine the binding constants and stoichiometry for each porphyrin-RNA binding event studied in this work. From the temperature dependences of the binding constants, we calculated the binding free energies, DeltaG(b), enthalpies, DeltaH(b), and entropies, DeltaS(b). For each RNA duplex, the binding enthalpy, DeltaH(b), is the most favorable for TEtOHPyP4 (an intercalator) followed by TAlPyP4 (an intercalator) and TMetAlPyP4 (an outside binder). On the other hand, for each duplex, external self-stacking of TMetAlPyP4 produces the most favorable change in entropy, DeltaS(b), followed by the intercalators TAlPyP4 and TEtOHPyP4. Thus, our results suggest that the thermodynamic profile of porphyrin-RNA binding may correlate with the binding mode. This correlation reflects the differential nature of molecular forces that stabilize/destabilize the two modes of binding-intercalation versus external self-stacking along the host duplex.

Kinetics↗
PubMed1995

Complete replication of poliovirus in vitro: preinitiation RNA replication complexes require soluble cellular factors for the synthesis of VPg-linked RNA.

Translation of poliovirion RNA in HeLa S10 extracts resulted in the formation of RNA replication complexes which catalyzed the asymmetric replication of poliovirus RNA. Synthesis of poliovirus RNA was detected in unfractionated HeLa S10 translation reactions and in RNA replication complexes isolated from HeLa S10 translation reactions by pulse-labeling with [32P]CTP. The RNA replication complexes formed in vitro contained replicative-intermediate RNA and were enriched in viral protein 3CD and the membrane-associated viral proteins 2C, 2BC, and 3AB. Genome-length poliovirus RNA covalently linked to VPg was synthesized in large amounts by the replication complexes. RNA replication was highly asymmetric, with predominantly positive-polarity RNA products. Both anti-VPg antibody and guanidine HCl inhibited RNA replication and virus formation in the HeLa S10 translation reactions without affecting viral protein synthesis. The inhibition of RNA synthesis by guanidine was reversible. The reversible nature of guanidine inhibition was used to demonstrate the formation of preinitiation RNA replication complexes in reaction mixes containing 2 mM guanidine HCl. Preinitiation complexes sedimented upon centrifugation at 15,000 x g and initiated RNA replication upon their resuspension in reaction mixes lacking guanidine. Initiation of RNA synthesis by preinitiation complexes did not require active protein synthesis or the addition of soluble viral proteins. Initiation of RNA synthesis by preinitiation complexes, however, was absolutely dependent on soluble HeLa cytoplasmic factors. Preinitiation complexes also catalyzed the formation of infectious virus in reaction mixes containing exogenously added capsid proteins. The titer of infectious virus produced in such trans-encapsidation reactions reached 4 x 10(7) PFU/ml. The HeLa S10 translation-RNA replication reactions represent an efficient in vitro system for authentic poliovirus replication, including protein synthesis, polyprotein processing, RNA replication, and virus assembly.

Cytidine Triphosphate↗