Synthesis Of RNA Polymerase Research Articles

Induction of RNA polymerase synthesis in Escherichia coli.

Studies on the rate of synthesis of the beta and beta' subunits of RNA polymerase in haploid strains of Escherichia coli K12 containing poorly-suppressed rif degrees am mutations provide conclusive evidence that synthesis of at least these two subunits is regulated.

Regulation of ribonucleic acid synthesis in Escherichia coli B/r: An analysis of a shift-up : III. Stable RNA synthesis rate and ribosomal RNA chain growth rate following a shift-up

Following a nutritional shift-up, both the fraction of functioning RNA polymerase engaged in the synthesis of stable RNA, ψ s, and the ribosomal RNA chain growth rate, c s, increase within five minutes to near their final post-shift steady-state values. The increase in these two parameters is sufficient to account completely for the observed sudden increase in the rate of RNA accumulation. This implies that the control of stable RNA synthesis following a shift-up does not involve an activation of an inactive reserve of RNA polymerase or a burst of RNA polymerase synthesis, but rather results from a shift of RNA polymerase-transcribing messenger RNA genes to ribosomal and transfer RNA genes along with some increase in the stable RNA chain growth rate.

T7 protein synthesis in F' episome-containing cells: assignment of specific proteins to three translational groups.

Synthesis of many T7 proteins is prevented in F' episome-containing cells. In order to quantitate the degree of inhibition, we measured the activity of several T7 proteins in extracts prepared from T7-infected F(-) and F' cells and cells containing F factors mutant in phage inhibition [F'(PIF(-)2A) and F'(PIF(-)2A,2B)]. In addition, we were able to assign specific T7 proteins to the three translational units previously defined by polyacrylamide gel analysis of T7 proteins made in F(-) and episome-containing cells. After T7 infection, the presence of the wild-type F' (PIF(+)) episome led to greater than 90% inhibition of T7 DNA polymerase (product of gene 5), T7 lysozyme (gene 3.5), and gene 10 capsid protein synthesis. Nearly normal amounts of T7 RNA polymerase (gene 1) were made in these cells. T7 infection of cells containing the mutant F' (PIF(-)2A) episome led to normal synthesis of T7 RNA polymerase and T7 DNA polymerase; T7 lysozyme was synthesized at 30% of the maximal level in these cells; T7 gene 10 capsid protein synthesis was inhibited by 90%, and T7 DNA synthesis was arrested in these cells. T7 infection of cells containing the mutant F' (PIF(-)2A,2B) episome led to synthesis of normal levels of the enzymes assayed.

The Effect of Metabolic Inhibitors and Hydroxylamine on the Immune Response in Mice to Mycobacterial Ribonucleic Acid Vaccines

Abstract Metabolic inhibitors were used in an attempt to determine the manner by which mycobacterial ribonucleic acid (myc. RNA) immunizes against tuberculosis. Inhibitors of protein synthesis (streptomycin sulfate, chloramphenicol, cycloheximide) and inhibitors of DNA-dependent RNA polymerase (rifampin) had no effect on the immune response of mice vaccinated with myc. RNA. All of these compounds except streptomycin sulfate, however, inhibited antibody formation under the same conditions. In contrast, inhibitors of deoxyribonucleic acid (DNA) synthesis significantly reduced the immune response. Five-fluoro-deoxyuridine (FUDR), which inhibits thymidine synthetase, when injected just before vaccination, reduced the immune response but had no effect if injected 18 hr after vaccination. These results indicated that early synthesis of DNA was important, and that DNA once formed was not affected by FUDR. Ethidium bromide, proflavine and chloroquine were each mixed with myc. RNA and injected into mice. Under these circumstances the immune response was significantly lowered. In addition, chloroquine was injected at various times and significant reduction in the immune response occurred only if it were injected 1 hr or later after vaccination. This compound binds with DNA, and shows again the necessity for the presence of DNA in the immune process. Actinomycin D injected at the time of vaccination or 1 hr after vaccination had no effect on the immune response. However, if actinomycin D was injected 18 hr after vaccination or injected daily for 4 days after vaccination, a marked reduction in the immune response occurred. These results indicated that actinomycin D, which inhibits RNA transcription may have inhibited the replication of myc. RNA. A mutogenic agent, hydroxylamine, also reduced significantly the immunogenic activity of myc. RNA. These results closely paralleled those obtained by other investigators in studies aimed at eliciting the mode of replication of oncogenic viruses. The replication of these RNA tumor viruses is unique in that DNA synthesis is required for viral RNA replication early after infection, and there is a requirement for RNA transcription. The similarity between the manner by which myc. RNA immunizes and RNA tumor viruses replicate is discussed, and a comparison also is made of the two diseases, tuberculosis and tumors formed by oncogenic viruses. These results strongly suggest that myc. RNA may immunize against tuberculosis by forming a DNA template in the host cell which then transcribes specific information for the replication of myc. RNA, and thereby transforms a normal cell into a specific immune cell. If correct, this would provide a new concept of the nature of cellular immunity of tuberculous infection.

Gene function and strand breaks in DNA of gamma-irradiated T7 bacteriophages.

SummaryThe effect of γ-irradiation on gene function in bacteriophage T7 was examined and compared with the alteration of the DNA structure. An early function tested was the synthesis of T7 specific RNA polymerase. Its relative radiosensitivity is 0·32 compared with the plaque-forming ability. The same relative sensitivity of 0·30 was observed for the inhibition of injection and for the formation of double-strand breaks. This observation confirms the hypothesis that, after infection with γ-irradiated phage, only DNA molecules with an intact double helix are injected into the host cell. The radiosensitivity of the synthesis of lysozyme, tested as late function, is 0·58 compared with plaque-forming ability.

Transcription of native and denatured DNA preparations by bacteriophage T3 induced RNA polymerase.

It is now well established that the expression of genetic information in bacteriophages, as well as in more complex organisms, is subject to temporal control; not all genes are expressed simultaneously. Early- and late–functioning cistrons have been distinguished in λ, T-even, and other bacteriophages. In these viruses, temporal regulation of gene expression seems to occur at the transcriptional level. At a certain time after infection, mRNA synthesis, originally specific for early phage proteins, is altered to produce RNA that directs the synthesis of late phage proteins. Evidence for this control mechanism has been reviewed (1,2). The change in transcription specificity leading to the synthesis of late mRNA may be achieved either by modification of the host RNA polymerase or by de novo synthesis of a new RNA polymerase coded by the viral genome. The modified or the new RNA polymerase can initiate RNA synthesis corresponding to late bacteriophage genes.

Synthesis of RNA polymerase in Escherichia coli B-r growing at different rates.

Abstract Polyacrylamide gel electrophoresis of unfractionated sodium dodecyl sulfate lysates of Escherichia coli B r has been used to investigate the synthesis of β and β′ subunits of RNA polymerase as a function of bacterial growth rate. In succinate (μ = 0.67 doublings/h), glucose (μ = 1.36 doublings/h) and glucose/amino acids (μ = 2.10 doublings/h) supplemented media, the fraction of [ 14 C]leucine-labeled β and β′ protein/total protein was found to be 1.05, 1.31 and 1.56%, respectively. Comparison of these values with recent estimates from this laboratory of the differential rate of synthesis of functioning RNA polymerase suggests an excess of total over functioning RNA polymerase. The significance of these data in reference to the regulation of RNA polymerase synthesis is discussed.

Biosynthesis of the β and β′ subunits of RNA polymerase in Escherichia coli

The amounts of the β and β′ subunits of the DNA-dependent RNA polymerase relative to the amount of total protein synthesized have been determined under a number of growth conditions in two strains of Escherichia coli. The results of these measurements have been expressed as the relative rate of synthesis of core RNA polymerase, α p, assuming the four constituent subunits (2α, 1β and 1β′) to be synthesized in equivalent amounts. This quantity, α p, was found not to vary greatly with the growth rate μ. For glucose-grown cells of E. coli B/r (μ = 1.5 doublings/h) α p = 1.4%, corresponding to about 7000 molecules of core RNA polymerase per cell. For slowgrowing cells the value obtained for α p is lower and for fast-growing cells somewhat 3 higher. The comparison of these values with the number of RNA polymerase molecules estimated to be actively engaged in RNA synthesis indicates that both slow- and fast-growing cells contain a surplus of RNA polymerase, if the catalytic unit is assumed to be the monomer of core RNA polymerase. In addition to the measurements of cells during balanced growth at various rates, α p has been determined during the transition from one growth rate to another and during synchronous growth. During a shift-up the rate of synthesis of polymerase follows closely the rate of total protein synthesis, α p being nearly constant for a period of twenty minutes after the shift. In a synchronously dividing culture of E. coli B/r, α p was seen to be fairly constant during two cycles of synchronous division. It appears that α p is rather insensitive to the effect of gene doubling during the cell cycle.

Synthesis of Mitochondrial RNA Polymerase

Synthesis of Mitochondrial RNA Polymerase

Effects of alpha-amanitin in vivo on RNA polymerase and nuclear RNA synthesis.

The fungal toxin α-amanitin given in vivo selectively inhibits for 10–60 min a nucleoplasmic type of RNA polymerase in rat liver but the synthesis in vivo of all species of nuclear RNA remains blocked for several hours.

Differential influence of spermidine on DNA directed enzyme synthesis in vitro.

Spermidine has a differential influence on T3 and T7 DNA-directed enzyme synthesis in vitro on the translational level. It increases the effectivity of synthesis of phage RNA polymerase, of ligase and of S-adenosylmethionine cleaving enzyme (SAMase), but decreases the effectivity of synthesis of lysozyme.

The early region of the DNA of bacteriophage T7.

RNA polymerase from Escherichia coli transcribes on T7 DNA in vitro and in vivo a polycistronic messenger RNA of 2.2 × 106 molecular weight. This mRNA contains the information for synthesis of T7‐phage RNA polymerase, ligase and lysozyme as was demonstrated by enzyme synthesis in vitro, using the RNA synthesized in vitro as template. Bystudying the kinetics of synthesis of these enzymes in vitro and the ultraviolet‐irradiation sensitivity of enzymesynthesis rates in vivo, the location of the genes on the DNA was estimated. Phage RNA polymerase is encoded within the first half of the mRNA molecule. The information for ligase ends at approximately 5500 nucleotides (1.7 × 106 mol wt.) and the information for lysozyme is close to the 3′‐end of the mRNA.The genes for T7 RNA polymerase and ligase are transcribed by the host RNA polymerase (early region), whereas the genes for endonuclease (gene 3) and for lysozyme are read by E. coli RNA polymerase and by T7‐phage RNA polymerase (early/late region). The DNA beyond the termination signal for host RNA polymerase is transcribed by T7‐phage RNA polymerase in the late phase (late region).Genetic mapping of mutants in the ligase and in the lysozyme genes confirm the data obtained by enzyme synthesis in vitro.

In vitro synthesis of T3 AND T7 RNA polymerase at low magnesium concentration

In vitro synthesis of T3 AND T7 RNA polymerase at low magnesium concentration

Gene Expression in Vitro from Deoxyribonucleic Acid of Bacteriophage T7

Abstract The in vitro synthesis of lysozyme and RNA polymerase directed by DNA of bacteriophage T7 starts with different lag periods after addition of the template. The difference in lag period before appearance of enzyme could be attributed to a difference in the time needed for transcription. Assuming that the difference in lag phase is caused by a difference in the distance between the promoter and the corresponding gene, and taking into account the average rates of transcription and translation, the locations of the polymerase gene and the lysozyme gene on the phage genome were estimated. The distance between the promoter and the distal end of the lysozyme gene corresponds to an RNA of the molecular weight 2.2 x 106. The polymerase gene is closer to the promoter. Transcription of T7 DNA by Escherichia coli RNA polymerase in vitro does indeed result in a polycistronic messenger RNA containing the information for synthesis of both RNA polymerase and lysozyme.

RNA polymerase synthesis in vitro directed by T7 phage DNA.

T7 RNA polymerase is synthesized in vitro, dependent on T7 DNA. The in vitro synthesized T7 polymerase has the characteristic properties: resistance to rifampicin and streptolydigin and the typical template specificity.

In vitro synthesis of a DNA dependent RNA polymerase coded on coliphage T7 genome.

In vitro synthesis of a DNA dependent RNA polymerase coded on coliphage T7 genome.

The effect of feeding with a tryptophan-free amino acid mixture on rat liver magnesium ion-activated deoxyribonucleic acid-dependent ribonucleic acid polymerase.

1. The Widnell & Tata (1966) assay method for Mg(2+)-activated DNA-dependent RNA polymerase was used for initial-velocity determinations of rat liver nuclear RNA polymerase. One unit (U) of RNA polymerase was defined as that amount of enzyme required for 1 mmol of [(3)H]GMP incorporation/min at 37 degrees C. 2. Colony fed rats were found to have a mean RNA polymerase activity of 65.9muU/mg of DNA and 18h-starved rats had a mean activity of 53.2muU/mg of DNA. Longer periods of starvation did not significantly decrease RNA polymerase activity further. 3. Rats that had been starved for 18h were used for all feeding experiments. Complete and tryptophan-deficient amino acid mixtures were given by stomach tube and the animals were killed 15-120min later. The response of RNA polymerase to the feeding with the complete amino acid mixture was rapid and almost linear over the first hour of feeding, resulting in a doubling of activity. The activity was still elevated above the starvation value at 120min after feeding. The tryptophan-deficient amino acid mixture produced a much less vigorous response about 45min after the feeding, and the activity had returned to the starvation value by 120min after the feeding. 4. The response of RNA polymerase to the feeding with the complete amino acid mixture was shown to occur within a period of less than 5min to about 10min after the feeding. 5. Pretreatment of the animals with puromycin or cycloheximide was found to abolish the 15min RNA polymerase response to the feeding with the complete amino acid mixture, but the activity of the controls was unaffected. 6. The characteristics of the RNA polymerase from 18h-starved animals and animals fed with the complete or incomplete amino acid mixtures for 1h were examined. The effects of Mg(2+) ions, pH, actinomycin D and nucleoside triphosphate omissions were determined. The [Mg(2+)]- and pH-activity profiles of the RNA polymerase from the animal fed with the complete mixture appeared to differ from those of the enzyme from the other groups, but this difference is probably not significant. 7. [5-(3)H]Orotic acid incorporation by rat liver nuclei in vivo was shown to be affected by the amino acid mixtures in a similar manner to the RNA polymerase. 8. The tryptophan concentrations of plasma and liver were determined up to 120 min after feeding with the amino acid mixtures. Feeding with the complete mixture produced a rapid increase in free tryptophan concentrations in both plasma and liver, but feeding with the incomplete mixture did not alter the plasma concentration. The liver tryptophan concentration increased at about 45min after feeding with the tryptophan-deficient diet. 9. There was a good correlation between the liver tryptophan concentration and RNA polymerase activity in all groups of animals. 10. It was concluded that the rat liver nucleus responded to an increase in amino acid supply by increased synthesis of RNA as a result of synthesis of RNA polymerase de novo. The correlation of tryptophan concentration and RNA polymerase activity appears to reflect the general amino acid concentration required to support hepatic protein synthesis and to produce new RNA polymerase. This new polymerase appears to differ from the basal RNA polymerase by its rapid synthesis and destruction, which may be a means of regulating RNA synthesis by the amino acid concentration in the liver.

New RNA polymerase from Escherichia coli infected with bacteriophage T7.

A new T7-specific RNA polymerase is found in T7 phage-infected cells. It is the product of T7 gene I and is physically and biochemically distinct from the host cell RNA polymerase. The synthesis of T7 RNA polymerase provides a direct explanation for the pleiotropic control of late T7 functions exerted by gene I.

Three cistrons in bacteriophage Qβ

Amber mutants of bacteriophage Qβ were isolated and classified into three groups by complementation tests. Physiological studies showed that the three cistrons of Qβ could be related to the three known genes of the f2 group of RNA phages. Gene I mutants do not produce either phage RNA polymerase or phage specific RNA in Su − hosts. Gene II mutants in Su − hosts produce defective phage particles which are lighter in density than viable phage particles. Gene III mutants do not grow on either Su-2 or Su-3 suppressor strains; they hyperproduce double-stranded RNA and viral RNA polymerase on these hosts. On Su − hosts they appeared to have a polar effect on the synthesis of phage RNA polymerase. None of the Qβ mutants lysed nonpermissive hosts.

Effect of 2-(alpha-hydroxybenzyl)-benzimidazole and guanidine on the uncoating of echovirus 12.

2-(α-HYDROXYBENZYL)-benzimidazole (HBB) and guanidine have been shown to inhibit selectively the multiplication of picornaviruses1. Both substances inhibit the appearance of a functional viral RNA polymerase2, the replication of viral RNA3,4, and the synthesis of viral coat protein4. While inhibition of synthesis of viral capsids seems to be a secondary phenomenon, probably a consequence of the failure of formation of viral RNA5, it is not clear whether the primary site of action of these compounds is principally the synthesis of viral RNA polymerase6,7. As to the early steps of virus-cell interaction, it was shown that virus adsorption and penetration remain unaffected by HBB and guanidine4,8, yet their effect on uncoating has not been determined. The period inhibitable by HBB and guanidine extends well into the exponential increase phase of the virus, thus covering the synthetic period of viral RNA polymerase and viral RNA, but it begins in the replication cycle at a time when synthesis of viral RNA polymerase and of viral RNA are not yet demonstrable3,4,8.