Enzyme patterns in mitochondria of eggs, liver, and skeletal muscle during larval development of Xenopus

The amebo-flagellate Naegleria gruberi contains three major RNA polymerase activities separable from whole cell extracts by DEAE-Sephadex column chromatography. One is resistant to the durg α-amanitin and the other two are sensitive to it. These separated activities were characterized and then examined during the differentiation of ameba to flagellate, which is dependent on RNA synthesis and which exhibits gross changes in RNA metabolism under the conditions employed. It was found that no detectable changes occurred qualitatively or quantitatively in these polymerases during differentiation. Care was taken to account for total enzyme activity in every fraction of the extraction procedure. It is concluded from the above observation that gross changes in RNA synthesis as well as differential gene activity can occur with absolutely no major fluctuations in the DNA-dependent RNA polymerases.     

Interaction of viral RNA with Escherichia coli. 3. Synthesis of poliovirus-specific RNA directed by isolated poliovirus RNA

Poliovirus RNA directs the synthesis of virus-specific RNA in E. coli as reported previously for poliovirus-induced double-stranded RNA. Synthesis of viral RNA can be followed by conversion of viral RNA into a double-stranded RNase-resistant state, by increase in infectivity and by hybridization of newly synthesized RNA to viral RNA. Virus-specific RNA synthesis occurs also in the presence of inhibitors of protein synthesis indicating that an enzyme is present in E. coli which can use RNA as a template.     

Polyadenylic acid synthesis activity of purified DNA-dependent RNA polymerase from Caulobacter

Characterization of purified DNA-dependent RNA polymerase (EC 2.7.7.6) of Caulobacter crescentus, strain CB15 has led to the conclusion that this enzyme catalyzes poly(A) synthesis in the absence of template. Poly(A) synthetase activity co-purifies with both holoenzyme and core polymerase on DNA-cellulose columns, and core polymerase purified to 98% homogeneity by glycerol gradient centrifugation is still capable of catalyzing poly(A) polymerization. Both RNA synthesis and poly(A) polymerization activities are sensitive to rifampicin. In addition, RNA polymerase purified from partially rifampicin-sensitive mutants exhibits the same partial sensitivity in vitro to the drug in the synthesis of RNA and poly(A). The enzyme used in these studies was prepared by a simple method which allows a high yield of pure RNA polymerase from large batches of exponential cells. The procedure includes high speed centrifugation of cell extracts, DEAE-cellulose column, DNA-affinity chromatography, and low salt glycerol gradient centrifugation. Holoenzyme can be resolved into core and sigma subunit by either DNA-cellulose chromatography or glycerol gradient centrifugation, and the latter step allows recovery of pure sigma factor.     

Replication of RNA viruses: specific binding of the Q RNA polymerase to Q RNA

The specific binding in vitro of the Qβ RNA polymerase to Qβ RNA has been detected by the formation of an enzyme-Qβ RNA complex that did not exchange bound RNA molecules and was not dissociated by 0.8 m NaCl. Formation of this nondissociating complex required GTP and two host protein factors, but not ATP, CTP, UTP, or Mg²⁺ ions. GDP, GMP, dGTP, ITP, and β,γ-methylene GTP did not replace GTP in the reaction. Complex formation at 0 °C was not observed, and the rates of the reaction at 30 °C and 25 °C were 41% and 23%, respectively, of the rate at 37 °C. The reaction occurred with intact Qβ RNA and with polycytidylic acid template but not with bacterial or other bacteriophage RNA. With limiting amounts of enzyme, the amount of Qβ RNA bound in the nondissociating complex was the same as the amount of [γ-³²P]GTP incorporated into nascent RNA chains, indicating a close relationship between complex formation and the initiation of RNA synthesis. The two reactions appear to be separate, however, because in the absence of Mg²⁺ ions, when complex formation occurred readily, no RNA synthesis could be detected either by incorporation of labeled substrate into acid-insoluble material or by formation of short RNA chains still attached to the enzyme. In the presence of factor protein and GTP, a maximum of one active enzyme molecule was bound per molecule of Qβ RNA template, as determined by a liquid polymer phase-separation procedure. These results suggest that formation of the nondissociating complex measures recognition by the Qβ RNA polymerase of a single Qβ RNA site utilized for the initiation of synthesis.     

Stimulation of fatty acid synthesis in vitro by gonadotrophin-induced testicular ribonucleic acid

RNA from testes of hypophysectomized rats treated with follicle-stimulating hormone and luteinizing hormone markedly stimulates in vitro the incorporation of acetate and malonate (as CoA derivatives) into polyunsaturated fatty acids. The system in vitro contains the components necessary for both protein and fatty acid synthesis. That the RNA is a hormone-induced messenger type that causes enzyme synthesis that then causes fatty acid synthesis is supported by the following observations: (1) the stimulation of RNA synthesis by follicle-stimulating hormone and luteinizing hormone is decreased by injection of the animals with actinomycin D; (2) puromycin in the system in vitro decreases the synthesis of polyunsaturated fatty acids; (3) the activity of the RNA preparation is destroyed by digestion with ribonuclease; in fact, the digest is inhibitory, which is a characteristic of messenger-RNA-mediated protein synthesis; (4) protein that might be denatured enzyme is virtually absent from the effective RNA preparations.     

Sub-nuclear fractionation. II. Intranuclear compartmentation of transcription in vivo and in vitro

DNA-dependent RNA polymerase activities were measured in subnuclear fractions obtained from rat liver by the procedure described in the preceding paper [14]. Most of the total nuclear enzyme was recovered in a form bound to chromatin with only small amounts as free enzyme in the nucleoplasm. The multiple eukaryotic RNA polymerases were resolved according to the endogenous template to which they were bound and which they continue to transcribe in vitro. The A and B forms of the enzyme were distinguished from each other by their differential sensitivities to α-amanitin, exogenous native and denatured DNA, thermal denaturation at 45 °, Mg²⁺ and Mn² ions, high ionic strength and by the binding of ${}^{14}\text{C-methyl}\text{-γ-}\text{amanitin}$. RNA polymerase B (α-amanitin-sensitive) was exclusively recovered in the nucleoplasmic and euchromatin fractions. RNA polymerase A was recovered in the dispersed nucleolar as well as in heterochromatin. By assaying in the presence of α-amanitin subnuclear fractions that had been pre-incubated at 45 °C a third enzyme (form C) was located exclusively in heterochromatin fractions. Only the euchromatin associated RNA polymerase B was capable of initiating the synthesis of new RNA chains in vitro on endogenous template at low ionic strength. Raising the ionic strength abolished initiation but accelerated chain elongation by this form of enzyme.When nuclear RNA was labelled in vivo, newly made RNA turned over rapidly in the nucleoplasm but accumulated in the euchromatin + membrane fraction. RNA in the nucleolar fraction accumulated gradually after a lag period, whereas a significant amount of rapidly-labelled nuclear RNA was recovered in the heterochromatin fractions. The distribution of RNA labelled in vivo compared with that of RNA polymerase activities suggested that RNA synthesized in vivo is rapidly translocated from its site of synthesis to some other sites within the nucleus.     

The sensitivity of RNA polymerases I and II from Novikoff hepatoma (N1S1) cells to 3''-deoxyadenosine 5''-triphosphate.

The synthesis of ribosomal precursor RNA in Novikoff hepatoma (N1S1) cells is very sensitive to cordycepin (3'-dA). The synthesis of hnRNA, however, is resistant to inhibition concentrations of 3'-dA that completely block the synthesis of 45S ribosomal RNA precursor. We have examined the RNA polymerases present in these cultured cells with regard to their sensitivity to cordycepin 5'-triphosphate (3'-dATP) in an effort to explain the differential inhibition of RNA synthesis observed in vivo. RNA polymerases I and II were characterized on the basis of their chromatographic behavior on DEAE-Sephadex, as well as the response of their enzymatic activities to ionic strength, the divalent metal ions Mn2+ and Mg2+, and the toxin alpha-amanitin. For both enzymes the inhibition of in vitro RNA synthesis by 3'-dATP was competitive for ATP. The km values for ATP and the K1 values for 3'-dATP for the two enzymes were quite similar. RNA polymerase II, the enzyme presumed responsible for hnRNA synthesis, was actually slightly more sensitive to 3'-dATP than RNA polymerase I, the enzyme presumed responsible for ribosomal precursor RNA synthesis. Similar data were obtained when the RNA polymerases were assayed in isolated nuclei. These results indicate that the differential inhibition of RNA synthesis caused by 3'-dA in vivo cannot be simply explained by differential sensitivity of RNA polymerases I and II to 3'-dATP.     

In vitro replication of cowpea mosaic virus RNA. II. Solubilization of membrane-bound replicase and the partial purification of the solubilized enzyme.

A method for the solubilization of membrane-bound Cowpea mosaic virus RNA replicase has been developed by bypassing the use of detergents. Solubilization has been achieved by washing the 31,000 x g-pellet containing the bound replicase with a Mg2+-deficient buffer. This procedure had several advantages as compared to treatments with nonionic or ionic detergents: (i) the solubilized enzyme was stable at 4 C, (ii) more than 80% of the replicase could be solubilized without loss of total enzyme activity, (iii) the replicase was rather selectively released resulting in a two- to threefold increase in specific activity per se, and (iv) most of the green color from chloroplast fragments present in the crude replicase fraction remained membrane bound resulting in only slightly colored preparations of solubilized enzyme. The solubilized replicase has been further purified by DEAE-Bio Gel column chromatography. RNA synthesis directed by the DEAE-purified enzyme was template dependent and proceeded at a linear rate for at least 9 h.     

Temporal changes in chromatin isolated from Friend virus-infected mouse spleens.

Differences noted in enzyme II directed RNA synthesis under varying salt conditions in nuclei isolated from uninfected and Friend virus (FV)-infected spleen cells, have been attributed to chromosomal modifications (Babcock and Rich 1973). This investigation was undertaken to determine if compositional changes occur in the chromatin of FV-infected spleens, which correlate with an altered rate of synthesis by enzyme II. A quantitative study of the chromatin constituents at various times after infection indicated that they vary in the same temporal manner as the rate of RNA synthesis in isolated nuclei. Relative to DNA, RNA, histone, and nonhistone protein reached a maximum at 14 days postinfection. This was followed by a gradual decrease during the remainder of the infection. Chromatin endogenous DNA-dependent RNA polymerase activity varied in the same manner, suggesting that RNA synthesis directed by enzyme II is modulated by chromosomal proteins.     

An improved method for the quantitative isolation of rat liver nuclear RNA polymerases.

Rat liver nuclear RNA polymerases exist in two functional states, one of which is active towards the endogenous chromatin template (engaged enzyme), while the other is inactive (free enzyme) (Yu, F.L. (1974) Nature 251, 344-346). This paper reports the direct separation of these two populations of RNA polymerases from isolated rat liver nuclei by a simple extraction procedure. It is estimated that as much as 50% of the total nuclear RNA polymerase activity in normal rat liver may exist in the form of the free enzyme. Evidence is also presented to indicate that the free enzyme activity is easily lost when the nuclear isolation procedure involves the use of an isotonic buffer medium, or when the isolated nuclei are subjected to sonication as is required for the solubilization of the nuclear RNA polymerases by the conventional method. Based on these new findings, it is proposed that nuclei be isolated directly in hypertonic sucrose and that the free enzyme be extracted before the nuclei are subjected to sonication to solubilize the engaged enzyme. This method circumvents the loss of the free RNA polymerase population and, as a result, the total yield of the nuclear RNA polymerases is greatly increased. The possible functional role of the free RNA polymerase in gene expression is discussed.     

Biosynthesis and turnover of carnitine acetyltransferase in rat liver

Male Wistar rats were fed on a diet with and without di(2-ethylhexyl)phthalate (DEHP) for 2 weeks. Carnitine acetyltransferase in the liver was increased about 100-fold by administration of DEHP. The results of in vivo experiments showed that the incorporation of L-[4,5-3H]leucine into the enzyme was 12-fold higher and the half-life of the labeled enzyme was elongated by a factor 4.6. The results of in vitro translation experiments with total hepatic RNA in a rabbit reticulocytelysate system and the results concerning the synthesis of the enzyme in isolated hepatocytes indicate that the translatable mRNA for the enzyme was increased upon administration of DEHP and that the enzyme is synthesized as a precursor (Mw = 69,000) larger than the mature enzyme (Mw = 67,500). RNA in the free polysomes directed the synthesis of the enzyme precursor five times more actively than RNA in membrane-bound polysomes.     

Convolution analysis of transcription by yeast DNA-dependent ribonucleic acid polymerase A. A mathematical method for studying ribonucleic acid chain elongation.

The rate of initiation of RNA synthesis catalysed by yeast RNA polymerase A on native calf thymus DNA decayed exponentially with a half-life of about 4.3 min. The rate constant for initiation was unaffected by preincubating the enzyme with DNA, or by decreasing the concentration of GTP 4-fold. The rate of RNA synthesis was constant for 15--20 min and then decreased. Each enzyme molecule made no more than one RNA molecule. In this situation, initiation, elongation and total RNA synthesis are related by a convolution integral. Solution of the convolution integral revealed that the rate of elongation was apparently biphasic. Analysis of the size of the RNA product showed that this biphasic profile arose because most but not all of the enzyme stopped RNA synthesis soon after initiation.     

Kinetic analysis of template-instructed and de novo RNA synthesis by Q beta replicase

The kinetics of template-free and template-instructed RNA synthesis by Q_β replicase were investigated. Template-instructed RNA synthesis has different growth rates in the exponential (excess enzyme) and the linear (excess template) phase of growth. In the absence of exogenous template, Q_β replicase synthesizes self-replicating RNA after an initial lag phase (“template-free” synthesis). The lag time can be determined by extrapolating the growth curve to the time of appearance of the first self-replicating strand. Growth rates in the exponential and linear phase, and especially the times of the lag phase for nucleotide incorporations under identical template-free conditions, show considerable scattering in contrast to the deterministic behavior of template-instructed synthesis. Evaluation of the kinetic data reveals that the time lag of template-free synthesis is strongly dependent on the concentration of the nucleoside triphosphate and the enzyme. The lag time is approximately inversely proportional to the powers 2.75 of the nucleotide and 2.5 of the enzyme concentration, respectively, both being lower limit values. The rate of template-instructed RNA synthesis is linearly proportional to the enzyme concentration and less than linearly proportional to the triphosphate concentration, in accordance with a substrate dependence of a Michaelis-Menten type of mechanism. The kinetic data cannot be reconciled with the proposition that template-free synthesis is due to low concentrations of templates present as impurities in the incorporation mixture and giving rise to autocatalytic RNA synthesis by a template-instructed mechanism. The data strongly favor the de novo mechanism proposed by Sumper &; Luce (1975).