Messenger Ribonucleic Acid Synthesis Research Articles

Regulation of maltodextrin phosphorylase synthesis in Escherichia coli by cyclic adenosine 3', 5'-monophosphate and glucose.

Cyclic adenosine 3', 5'-monophosphate (AMP) stimulates maltodextrin phosphorylase synthesis in Escherichia coli cells induced with maltose. A maximal effect occurs at 2 to 3 mM cyclic AMP. The action of cyclic AMP is specific, inasmuch as adenosine triphosphate, 3'-AMP, 5'-AMP, adenosine, and dibutyryl cyclic AMP are inactive. Glucose, alpha-methyl glucoside, 2-deoxyglucose, and pyridoxal 5'-phosphate repress maltodextrin phosphorylase synthesis. This repression is reversed by cyclic AMP. The action of cyclic AMP appears to be at the transcriptional level, since cyclic AMP fails to stimulate phosphorylase production in induced cells in which messenger ribonucleic acid synthesis has been arrested by rifampin or by inducer removal. The two other enzymes involved in the metabolism of maltose, amylomaltase and maltose permease, are also induced in this strain of E. coli and affected by glucose and cyclic AMP in a manner similar to phosphorylase.

Rate of Ovalbumin Messenger Ribonucleic Acid Synthesis in the Oviduct of Estrogen-primed Chicks

Abstract The concentration of translatable ovalbumin messenger RNA in the tubular gland cells of the chicken oviduct was determined from estimates of relative rate of ovalbumin synthesis, polysome concentration, size of ovalbumin-synthesizing polysomes, DNA concentration, and percentage of tubular gland cells in the oviduct. During secondary stimulation with estrogen, the concentration of ovalbumin messenger RNA rose from undetectable levels to approximately 70,000 molecules per tubular gland cell after 5 days when a steady state was reached. The initial rate of ovalbumin messenger RNA accumulation was about 22 molecules per min per tubular gland cell. Nearly all the translatable ovalbumin messenger RNA was localized in polysomes, even at very early times after hormonal stimulation, suggesting that there is no free pool of untranslated messenger. The half-life of ovalbumin messenger RNA was estimated to be about 24 hours by labeling the RNA in vivo followed by subsequent purification and determination of its specific activity. Two other potential approaches for determining messenger half-life were shown to be unsuitable. Using the half-life value of 24 hours, the steady state rate of ovalbumin messenger RNA synthesis was about 34 molecules per min per tubular gland cell. Based on theoretical values for the maximum rate of messenger RNA transcription in eukaryotes, one ovalbumin gene per haploid genome is sufficient to maintain the observed rate of ovalbumin messenger RNA synthesis.

Messenger ribonucleic acid synthesis and degradation in Escherichia coli during inhibition of translation.

Various aspects of the coupling between the movement of ribosomes along messenger ribonucleic acids (mRNA) and the synthesis and degradation of mRNA have been investigated. Decreasing the rate of movement of ribosomes along an mRNA does not affect the rate of movement of some, and possibly most, of the RNA polymerases transcribing the gene coding for that mRNA. Inhibiting translation with antibiotics such as chloramphenicol, tetracycline, or fusidic acid protects extant mRNA from degradation, presumably by immobilizing ribosomes, whereas puromycin exposes mRNA to more rapid degradation than normal. The promoter distal (3') portion of mRNA, synthesized after ribosomes have been immobilized by chloramphenicol on the promoter proximal (5') portion of the mRNA, is subsequently degraded.

Derepression of Anthranilate Synthase in Purified Minicells of Escherichia coli Containing the Col- trp Plasmid

Purified minicells of Escherichia coli K-12 containing the plasmid Col-trp(+) or Col-trpA2 could be derepressed for the synthesis of anthranilate synthase, the first enzyme encoded in the trp operon. Non-plasmid-containing, deoxyribonucleic acid-deficient minicells could not be derepressed. Derepressed enzyme synthesis was initiated by l-tryptophan starvation. The kinetics of derepression were studied with minicells containing the Col-trpA2 plasmid. The derepression curves were biphasic with a rapid initial rate of enzyme synthesis followed by a slower rate of synthesis. The presence of l-tryptophan (20 to 50 mug/ml) or chloramphenicol (200 mug/ml) abolished enzyme synthesis. The presence of rifamycin SV (280 mug/ml) partially inhibited enzyme synthesis after at least 3.5 min of exposure. The ratio of minicell-to-cell synthetic capacity was 1:2.4 when compared on the basis of derepressed enzyme activity per unit cell volume. This work demonstrates that plasmid-containing minicells are capable of considerable functional protein and messenger ribonucleic acid synthesis and that the regulation of at least the trp operon is similar in minicells to that observed in cells.

Requirement of adenosine-3',5'-cyclic monophosphate for L-arabinose isomerase synthesis in Escherichia coli.

Adenosine-3', 5'-cyclic monophosphate (cyclic AMP) is essential for the synthesis of l-arabinose isomerase in Escherichia coli. Cyclic AMP appears to be required for the transcription of deoxyribonucleic acid into messenger ribonucleic acid (RNA), since the enzyme synthesis is not observed in induced cells to which cyclic AMP is added after messenger RNA synthesis is arrested by rifampin or after inducer removal.

Role of adenosine 3',5'-cyclic monophosphate and its specific binding protein in the regulation of D-serine deaminase synthesis.

Adenosine 3',5'-cyclic monophosphate (cyAMP) and the cyAMP-binding protein are necessary for efficient induction of d-serine deaminase (Dsdase) synthesis in dsdC(+)dsdO(+) and dsdC dsdO(+) strains of Escherichia coli K-12, and for constitutive synthesis in dsdC dsdO(+) strains. Neither is required in dsdO strains, confirming previous indications that in dsdO mutants Dsdase synthesis is not subject to catabolite repression control. Since efficient Dsdase synthesis in dsdC(+) and dsdC strains that are dsdO(+) requires the cyAMP-binding protein, which acts at the level of transcription in other systems, it is concluded that catabolite repression acts at the level of transcription of the Dsdase structural gene. Since catabolite repression is reversed by the inducer, d-serine in dsdC dsdO(+) strains, it is concluded that induction also acts at the level of transcription in this system. The dsdC strains were found to be much more sensitive to induction by d-serine than are dsdC(+) strains under conditions of catabolite repression, whether the repression was caused by glucose or by loss of ability to form cyAMP or cyAMP-binding protein. This suggests that a d-serine-dsdC complex may be able to replace partially the cyAMP:cyAMP-binding protein action in initiation of Dsdase messenger ribonucleic acid synthesis-a positive control effect.

Control of the Synthesis of T4 Phage Deoxynucleotide Kinase Messenger Ribonucleic Acid in Vivo

Abstract The regulation of the synthesis of messenger RNA of T4 phage-specific deoxynucleotide kinase has been studied in vivo by assay of mRNA for its capacity to direct the synthesis of deoxynucleotide kinase in vitro. The deoxynucleotide kinase mRNA first appears at 2 min after infection and increases in amount until 8 min. Thereafter the messenger activity decreases. The appearance of deoxynucleotide kinase mRNA is blocked when chloramphenicol is added before or immediately after infection. When added at 8 min the normal levels of kinase mRNA were observed at times later in the infection period. However, the addition of chloramphenicol at 3 min after infection results in the production of an exaggerated amount of deoxynucleotide kinase mRNA, which keeps increasing until at least 30 min after infection. The high level of mRNA formed in the presence of chloramphenicol could reflect the absence of an agent or agents required for the termination of the synthesis of this mRNA. This conclusion was reached from experiments with the inhibitor of RNA synthesis, rifampicin. When added to cultures at a time late in the infection period, rifampicin caused an immediate drop in the level of kinase mRNA. At this time the level of this mRNA is therefore a balance between synthesis and degradation. When similar experiments were carried out to study the level of mRNA for α-glucosyltransferase, cultures receiving chloramphenicol at 3 min after infection and rifampicin at 11 and 17 min did not show a drop in the level of mRNA for this enzyme. There is thus a difference in the stability of the two types of mRNA in regard to their degradation by bacterial nucleases when the infection was carried out with chloramphenicol added at 3 min. In the absence of chloramphenicol both kinds of mRNA underwent degradation. The overproduction and the delay of shutoff of the synthesis of deoxynucleotide kinase mRNA are also seen when nonpermissive Escherichia coli K-12 is infected with T4 DO mutants or when DNA synthesis is inhibited by fluorodeoxyuridine. The infection with an MD mutant does not result in the same effect as that observed with the DO mutants. We favor the hypothesis that both new T4 protein (or proteins) and continuous T4 DNA replication are necessary for the proper termination of synthesis of delayed-early T4 RNA.

Transcription during the development of bacteriophage phi 29: production of host- and phi 29-specific ribonucleic acid.

The synthesis of ribonucleic acid (RNA) during development of the virulent Bacillus subtilis bacteriophage phi29 has been analyzed. Transcription of host deoxyribonucleic acid (DNA) continues at the preinfection rate throughout the latent period of viral growth. RNA-DNA hybridization was used to show that host messenger RNA synthesis continues late into the phage lytic cycle. Amino acid-labeling experiments show that this RNA is continuously used to produce protein. Ribosomal RNA production is not inhibited by phage infection. Small quantities of phage-specific RNA first appear between min 6 and 9 after infection. This RNA is made exclusively from one of the phi29 DNA strands. At 12 min postinfection, when phage DNA replication commences, large quantities of viral RNA start to be synthesized. This RNA appears to be transcribed from both strands of phi29 DNA. Studies with rifamycin and rifamycin-resistant host strains showed that the production of all phage phi29-specific RNA requires those components of the host RNA polymerase which are sensitive to this antibiotic. Thus, phage phi29 does not stop transcription of host DNA and may produce only one element for regulation of transcription of its own DNA. These findings may reflect the limited amount of genetic information carried by this phage.

Isoleucine and threonine can prolong protein and ribonucleic acid synthesis in pyridoxine-starved mutants of Escherichia coli B.

Pyridoxineless mutants of Escherichia coli B stopped incorporation of nucleosides into trichloroacetic acid-insoluble material about 40 to 60 min after pyridoxine starvation was initiated, whereas incorporation of amino acids (measured the same way) slowed but did not stop for several hours. Both these incorporations and cell density were increased most effectively by the presence of either threonine or isoleucine. Arginine, glutamate, histidine, methionine, tryptophan, and tyrosine also caused significant but less dramatic increases. Inducibility of beta-galactosidase continued beyond the point where nucleic acids appeared to stop their synthesis, suggesting that messenger ribonucleic acid synthesis continued beyond ribosomal ribonucleic acid synthesis. This inducibility was also increased by isoleucine and threonine. The overall results suggest that the threonine-isoleucine biosynthetic pathway is the most sensitive to starvation for pyridoxine.

Synthesis and decay of messenger ribonucleic acid from the lactose operon of Escherichia coli during amino-acid starvation

Synthesis and decay of messenger RNA from the lac operon of Escherichia coli were studied during amino-acid starvation and normal growth in suA + suA − and rel + rel − strains. At least 35% of the β-galactosidase message length is transcribed during leucine starvation, but there is no evidence that full-length molecules are made during either leucine or tryptophan deprival. In contrast, trp operon mRNA is completed during the same starvations. In both a stringent or relaxed strain βG message is inactivated at the same rates during starvation and normal growth. In normal growth the suA product does not affect the inactivation rates of either βG or transacetylase messages or the chemical loss of lac or total mRNA. However, it does account for 20 to 40% of their decay during leucine starvation. During growth TA message is inactivated two or three times faster than βG message. In starvation TA message is not made, but capacity to complete it and to make TA can be measured after re-addition of leucine. This capacity is lost at a much slower rate than during normal growth and at approximately the same rate as is capacity for βG synthesis. These results suggest: (i) a coupling of transcription to translation in the lac operon during amino-acid starvation, (ii) common targets for loss of capacity of both βG and TA messages on the operator-proximal part of lac mRNA, (iii) a role of the suA product in decay of lac mRNA during starvation and (iv) close to normal decay rates of the incomplete messages even though peptide synthesis is only 7% of normal.

The effect of antibiotics on the in vivo synthesis of messenger ribonucleic acid from the lactose operon of Escherichia coli.

Lac transcription is rapidly reduced to 3–5% of the original value in E. coli cells treated with antibiotics which block protein synthesis in general. Chloramphenicol (CM) exerts the most rapid, streptomycin (Sm) an intermediate, puromycin a slower effect. Cyclic AMP relieves about 15% of the CM-, 20% of the Sm-, and 40–60% of the puromycin effect indicating an increasing role of catabolite repression in the reduction of lac transcription. The remaining effects are due to the arrest of ribosomes on the nascent newly synthesized mRNA (CM and Sm) and probably the release of ribosomes from the RNA after termination of peptide synthesis (puromycin). In both cases mRNA unprotected by ribosomes is synthesized which either becomes rapidly degraded or hinders the further progression of RNA polymerase. Experiments with the polarity suppressor suA+ argue for the rapid degradation. Both, CM and Sm, reduce the total mRNA synthesis of E. coli to only 70%, while lac transcription is reduced to 15–20% (in the absence of catabolite repression). A possible reason for this difference is discussed.

Accumulation of 70S monoribosomes in Escherichia coli after energy source shift-down.

When Escherichia coli is shifted from glucose-minimal to succinate-minimal medium, a transient inhibition of protein synthesis and a time-dependent redistribution of ribosomes from polysomes to 70S monosomes occurs. These processes are reversed by a shift-up with glucose. In a lysate made from a mixture of log-phase and down-shifted cells, the 70S monosomes are derived solely from the down-shifted cells and are therefore not produced by polysome breakage during preparation. This conclusion is supported by the absence of nascent proteins from the 70S peak. The monosomes are not dissociated by NaCl or by a crude ribosome dissociation factor, so they behave as "complexed" rather than "free" particles. When down-shifted cells are incubated with rifampin to block ribonucleic acid (RNA) synthesis, the 70S monosomes disappear with a half-life of 15 min. When glucose is also added this half-life decreases to 3 min. The 70S particles are stable in the presence of rifampin when chloramphenicol is added to block protein synthesis. We interpret these data to mean that the existence of the 70S monosomes depends on the continued synthesis of messenger RNA and their conversion to free ribosomes (which dissociate under our conditions) is a result of their participation in protein synthesis. Finally, a significant fraction of the RNA labeled during a brief pulse of (3)H-uracil is found associated with the 70S peak. These results are consistent with the hypothesis that the 70S monosomes are initiation complexes of single ribosomes and messenger RNA, which do not initiate polypeptide synthesis during a shift-down.

Dissociation of Lac Messenger Ribonucleic Acid Transcription from Translation During Recovery from Inhibition of Protein Synthesis

Studies were made of the synthesis of Lac messenger ribonucleic acid (mRNA) by Escherichia coli in the absence of protein synthesis and of the coupling of transcription of lac operon to translation. Lac mRNA was not synthesized in the presence of chloramphenicol, and its synthesis steadily decreased during K(+) deprivation and treatment with puromycin. Since under these conditions total mRNA synthesis is not inhibited it is suggested that the control of Lac mRNA is distinct from that which regulates total mRNA synthesis. Lac mRNA synthesized during recovery from K(+) starvation or from chloramphenicol inhibition is not translated into functional enzyme, suggesting translational control over beta-galactosidase synthesis.

Inhibition of early vaccinia virus ribonucleic acid synthesis in interferon-treated chicken embryo fibroblasts.

Pretreatment of chicken embryo cells with homologous but not heterologous interferon inhibits the synthesis of vaccinia virus early messenger ribonucleic acid (mRNA). This inhibition is seen in the presence of cycloheximide, i.e., in the absence of protein synthesis, suggesting that the virion-bound transcriptase may be the target of the antiviral activity associated with interferon treatment. The inhibition of viral mRNA synthesis is dependent on the amount of chicken interferon used. The nonviral interferon inducer, polyriboinosine.polyribocytidine, similarly inhibits viral early mRNA synthesis in a dose-specific manner. The helical polynucleotide polydeoxyinosine.polyribocytidine, which is not an effective interferon inducer in chicken embryo cells, has no effect on viral ribonucleic acid synthesis.

Regulation of Gal Messenger Ribonucleic Acid Synthesis in Escherichia coli by 3',5'-Cyclic Adenosine Monophosphate

DNA-RNA hybridization experiments show that transcription of the gal operon is regulated by cyclic AMP as well as inducer. Cyclic AMP stimulates gal mRNA synthesis and does not affect its half-life. Mutants unable to make cyclic AMP synthesize gal mRNA only after the nucleotide is added. Mutants lacking a protein required for cyclic AMP action do not make gal mRNA. We conclude that lac and gal transcription is similarly controlled by cyclic AMP.

Synthesis of messenger ribonucleic acid after bacteriophage T1 infection.

Synthesis of host-specific and phage-specific messenger ribonucleic acid (mRNA) was studied in bacteria infected by unmodified (T1 . B) or modified [T1 . B(P1)] bacteriophage T1. In a "standard" infection of Escherichia coli B by T1 . B (no host-controlled modification involved), the rate and amount of T1 mRNA synthesis was intermediate between those values reported for infections by a virulent phage such as T4 or a temperate phage such as lambda. The initial rate of mRNA synthesis was slightly increased after T1 . B(P1) infection of E. coli B in comparison with T1 . B infection of the same host. Little or no phage mRNA synthesis could be detected in T1 . B infection of E. coli B(P1). Phage mRNA synthesis in T1 . B(P1)-infected E. coli B(P1) cells was approximately the same in amount as that seen in T1 . B(P1) infection of E. coli B. Synthesis of host-specific mRNA continued throughout the latent period in all infections studied. However, the enzyme beta-galactosidase could not be induced, except after T1 . B infection of E. coli B(P1). In an attempt to understand the apparent differences in mRNA synthesis after infection of E. coli B by phages T1 . B or T1 . B(P1), the effect of altered T1 deoxyribonucleic acid (DNA) methylation on mRNA synthesis was studied. Methyl-deficient T1 DNA, made in cells infected with ultraviolet-irradiated phage T3, inhibited (14)C-uridine incorporation more strongly than normal T1. One passage of methyl-deficient T1 through E. coli B restored uracil incorporation rates to those seen with ordinary T1. This suggests that methylation of T1 DNA can influence the rate of phage mRNA synthesis. However, attempts to relate the difference in mRNA synthesis seen between T1 . B and T1 . B(P1) in E. coli B to the activity of the P1 modification gene were not conclusive.

Inhibition of Poxvirus replication by streptovaricin.

A component of streptovaricin complex inhibits the replication of poxvirus. To be effective, the inhibitor must be introduced early in the replication cycle; it appears to inhibit early messenger ribonucleic acid synthesis by viral cores, thus interfering with all subsequent events. Neither of the two major components of the complex, streptovaricin A or C, was the active component.

Effects of amino acid starvation on constitutive tryptophan messenger ribonucleic acid synthesis

In both R −trp stringent ( rel + ) and R −trp relaxed ( rel − ) strains of Escherichia coli, starvation for arginine, leucine, histidine or isoleucine resulted in a marked decrease in the level of tryptophan messenger RNA as assayed by competitive hybridization experiments with DNA from a non-defective tryptophan transducing phage ( φ80pt190). Using the level of tryptophan messenger RNA in a wild-type genetically repressed strain as a measure, this was found to be reduced from roughly ten times the repressed level to 1.5 times this level in the case of starvation for arginine, leucine or isoleucine and three times this amount in the case of histidine starvation. Tryptophan messenger RNA synthesis, as reflected in the rate of labeling by [ 3H]uridine, is also greatly decreased by amino acid starvation in both strains. The results show that tryptophan messenger RNA is subject to a type of amino acid control distinct from the rel + ( RC str ) system described by Stent & Brenner (1961).

Regulation of Lac Messenger Ribonucleic Acid Synthesis by Cyclic Adenosine 3',5'-Monophosphate and Glucose

Abstract Cyclic adenosine 3',5'-monophosphate reverses the glucose repression of β-galactosidase synthesis in Escherichia coli induced for enzyme synthesis. In this report, we describe hybridization assays for the measurement of synthesis rates of lac messenger RNA and demonstrate that cyclic adenosine 3',5'-monophosphate and glucose alter rates of mRNA production in proportion to their effects on β-galactosidase synthesis. Thus we confirm earlier indirect measurements which indicate that control of β-galactosidase synthesis by cyclic adenosine 3',5'-monophosphate and glucose occurs at the level of transcription. In addition, we show that these effects on lac mRNA synthesis are observed in cultures incapable of enzyme synthesis and that glucose does not affect the rate of degradation of lac mRNA.

Possible selective genetic controls in synthesis of messenger ribonucleic acid by means of "paired" histones along the wide groove of the double helix of deoxyribonucleic acid.

Research Article| April 01 1970 Possible selective genetic controls in synthesis of messenger ribonucleic acid by means of ‘paired’ histones along the wide groove of the double helix of deoxyribonucleic acid S Lewin S Lewin Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1970) 117 (2): 19P–20P. https://doi.org/10.1042/bj1170019Pb Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation S Lewin; Possible selective genetic controls in synthesis of messenger ribonucleic acid by means of ‘paired’ histones along the wide groove of the double helix of deoxyribonucleic acid. Biochem J 1 April 1970; 117 (2): 19P–20P. doi: https://doi.org/10.1042/bj1170019Pb Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1970 The Biochemical Society1970 Article PDF first page preview Close Modal You do not currently have access to this content.