Post-transcriptional Steps Research Articles

Two complementation classes of T200 (Ly-5) glycoprotein-negative mutants.

Mutants of murine lymphomas have been isolated which express less than 1 percent of wild-type levels of T200 (Ly-5) glycoprotein on their cell surface but express wild-type levels of other cell-surface antigens tested. These mutants define two genetic complementation classes. The Class A mutant does not synthesize detectable amounts of T200 glycoprotein and is a mutant in either the structural gene coding for the T200 glycoprotein or in a gene acting in cis position to regulate this structural gene. The Class B mutant synthesizes very low levels of T200 glycoprotein and is most simply interpreted as a mutant in a gene acting at some post-transcriptional step necessary for expression of the T200 glycoprotein on the cell surface.--The isolation of T200- mutants by cytotoxic immunoselection suggests that this method is a general way of studying the biosynthesis, regulation and function of cell-surface molecules.

Interferon-mediated inhibition of mouse mammary tumour virus (type B) and mouse leukaemia virus (type C) in the same culture.

The sensitivity of mouse mammary tumour virus (MMTV) and murine leukaemia virus (MuLV) to interferon was tested by investigating the effect of interferon in cultures of mouse mammary tumour cells simultaneously synthesizing the two viruses. The dose-response curves showed that the extracellular production of both viruses by these cells was equally inhibited by interferon treatment. In contrast, the intracellular steady-state concentration of virus-specific RNA of both MMTV and MuLV was not significantly affected in these cells. This was the case regardless of whether total cellular RNA or polysomal poly(A)-containing RNA was examined. These results are consistent with the suggestion that interferon-caused inhibition is related to a host-mediated effect on a post-transcriptional step in virus synthesis.

Enzyme induction in a temperature-sensitive cell cycle mutant of Chinese hamster fibroblasts.

A temperature-sensitive (ts) cell cycle mutant of Chinese hamster fibroblasts with a block in G1 was investigated. Attention was on the expression of the activity of three enzymes: ornithine decarboxylase (ODC) S-adenosylmethionine decarboxylase (SAMDC), and thymidine kinase (TK). ODC and SAMDC activities are normally induced in the middle of, or late in, the G1 phase, while TK activity starts to appear at the G1/S boundary. In the ts mutant released from serum starvation at the nonpermissive temperature (40.8 degrees C), we find no effect on the expression of SAMDC activity, a significantly reduced level of ODC activity compared to the control at the permissive temperature (34 degrees C), and no induction of TK activity. Results presented here and in a previous publication (Landy-Otsuka and Scheffler, '78) suggest that the decrease in ODC activity is due to an effect of the nonpermissive temperature on a post-transcriptional step, possibly a very rapid inactivation of the enzyme. The absence of TK activity, on the other hand, appears to be due to a block in transcription at the nonpermissive temperature.

Autogenous and post-transcriptional regulation of Escherichia coli RNA polymerase synthesis in vitro.

The in vitro synthesis of Escherichia coli RNA polymerase β and β′ subunits is repressed by either holoenzyme or α2β complex. The level of mRNA synthesized in this system, from the genes encoding ribosomal protein L7/12 (rplL) and RNA polymerase ββ′ subunits (rpoBC) was separately determined by DNA-RNA hybridization using the plasmids, pLOO and pLBC, each containing rplL and rplL plus rpoBC, respectively, as DNA probes. Although the synthesis of β and β′ polypeptides was repressed by exogenous addition of the autorepressors, the amount of rpoBC mRNA increased in parallel with the total RNA synthesis. Thus, the relative concentration of rpoBC mRNA stayed at a constant level. Based on the simultaneous determination of both protein and mRNA, it was concluded that the autogenous regulation of RNA polymerase β and β′ subunit synthesis operates post-transcriptionally, presumably at the translational level. Similarly, glycerol inhibition of β and β′ subunit synthesis was also found to be operative at post-transcriptional step(s). In contrast, the rifampicin induction of β and β′ subunit synthesis is due to both the increased transcription of rpoBC genes and the relaxation of the autogenous repression.

Autogenous and post-transcriptional regulation of RNA polymerase synthesis

The regulation of gene expression was studied, for the Escherichia coli rpoBC operon, which includes the genes, rpoB and rpoC, for the beta and beta subunits of RNA polymerase, and rplJ and rplL, for the two proteins, L10 and L7/12, of the 50S ribosome. The gene organization agrees well with the accumulated observations indicating the coordinate synthesis of RNA polymerase and ribosomes under various growth conditions for wild-type E. coli cells. On the other hand, the differential regulation of the two essential components observed under restrictive growth conditions, after addition of various drugs or with certain mutants, in particular those carrying mutations in the RNA polymerase genes, was found to take place through two novel regulation systems: The transcriptional termination at an internal attenuation site and the two autogenous and posttranscriptional controls, being specific for the two ribosomal protein genes and the two RNA polymerase subunit genes, respectively. The majority of the transcription initiated from the promoter rpoP beta terminates at an attenuator site between the promoter-proximal rplJL and the promoter-distal rpoBC genes. The frequency of the attenuation seems to control the relative level of RNA polymerase synthesis to that of ribosomes. The expression of rpoBC genes is subject to an autogenous regulation, in which both RNA polymerase holoenzyme and alpha 2 beta complex function as regulatory molecules with repressor activity. The autogenous regulation was found to operate at post-transcriptional step(s), probably at the level of translation. During the study on the regulation of RNA polymerase synthesis, we noticed that the rpoBC operon contained another autogenous regulation circuit, in which the synthesis of L10 and L7/12 was specifically repressed by the L10-L7/12 complex. Molecular mechanisms and physiological meanings of the novel regulations are discussed.

Induction of tyrosine aminotransferase by dibutyryl cyclic-AMP in synchronized hepatoma cells

In Hepatoma Tissue Culture (HTC) cells induction of tyrosine aminotransferase (TAT) by dibutyryl cAMP (DBcAMP) is regulated at some posttranscriptional step. In synchronized HTC cells TAT can be induced by DBcAMP in late G 1 and in the S phase of the cell cycle only.

Inhibition of vaccinia virus growth by zinc ions: effects on early RNA and thymidine kinase synthesis

Accumulation of thymidine kinase activity in vaccinia virus-infected cells was severely inhibited by zinc ions if the drug was added within 1 h postinfection. If added later, zinc ions had no effect on the enzyme synthesis. A fraction of RNA which is normally synthesized in infected cells, was missing from a proper part of the gradient if the cells were treated with zinc ions within 1 h postinfection (as has been shown by cosedimentation of pulse-labeled RNAs in isokinetic gradients). It is suggested that a transcriptional (or posttranscriptional) step is involved in zinc-caused inhibition of vaccinia virus growth.

Interferon inhibits C-type virus at a posttranscriptional, prerelease step.

The mode of action of interferon in JLSV 5-cells, chronically infected with Rauscher murine leukemia virus (MLV), was studied by examining the fate of preexisting labelled viral RNA in interferon-treated cells and by determining the infectivity/physical particle ratio of cell-associated and extracellular virus. Interferon added together with 3H-uridine inhibited the production of labelled virus particles even when it was only allowed to act after all viral RNA synthesis had been stopped by actinomycin D. This indicated that the interferon-induced antiviral state primarily functions at a posttranscriptional step. When interferon was given after a 3H-uridine pulse label and arrest of label incorporation by glucosamine and unlabelled uridine, it prevented a portion of the preexisting radioactive RNA from occurring in extracellular particles. However, part of the labelled viral RNA had reached a stage beyond which interferon could not prevent it from occurring in extracellular virus particles. The notion that interferon primarily affects release of fully assembled and enveloped MLV particles may be eliminated: interferon-treatment did not affect the release of particle-bound reverse transcriptase in cells treated with cycloheximide after the antiviral state had been established. It was confirmed that interferon-treated JLSV 5-cells contained an increased number of virus particles associated with the cell membrane. However, these particles were found to have a reduced infectivity compared to those associated with control cells, thus confirming the view that virions produced by interferon-treated cells are defective; perhaps lacking in certain components.

Regulation of glucose carriers in chick fibroblasts.

The derepression of glucose transport initiated by removing glucose from the incubation medium requires both protein and RNA synthesis. The synthesis and accumulation of putative mRNA for the carrier protein(s) can be demonstrated by inhibiting protein synthesis with cycloheximide (2 microgram/ml). Release from inhibition with simultaneous addition of actinomycin D (1-5 microgram/ml) results in a burst of carrier synthesis that achieves virtually maximal derepression in 4-6 h. An external energy source provided by a "nonrepressive" sugar (D-fructose, D-xylose) or by pyruvate is required to accomplish carrier synthesis. Previous failure to demonstrate mRNA accumulation was due to the depletion of energy in the starved cells. Glucose acts as a repressor at a posttranscriptional step, probably at the level of turnover of formed carrier. The protection of formed carrier in the absence of glucose and by inhibitors of protein synthesis even in the presence of glucose has encouraged conjecture that a protease is activated by a metabolic product of glucose that is analogous to a corepressor. The glucose metabolite either activates the protease by direct interaction with it or alters the conformation of the carrier to expose a critical region to protease attack. Indeed the regulation of carrier density in the membrane of chick fibroblasts may be achieved entirely by carrier inactivation, the rate of which is a function of glucose concentration in the culture medium.

Effect of cold exposure on phosphoenolpyruvate carboxykinase (GTP) activity and cyclic AMP concentration in livers of starved rats role of glucocorticoids

The effect of cold exposure (5 degrees C) on the concentration of cyclic AMP and on the activity of phosphoenolpyruvate carboxykinase (GTP: oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32) was investigated in the liver of intact and adrenalectomized starved rats. Intact starved rats responded to cold exposure with a large increase in both the concentration of hepatic cyclic AMP and the activity of phosphoenolpyruvate carboxykinase above the starvation level. Adrenalectomy did not impair the cold-induced maximum elevation of cyclic AMP but totally prevented the response of the enzyme to cold. Yet, this response was completely restored by hydrocortisone treatment, while the steroid per se had no effect on enzyme activity. In isolated perfused livers of intact starved rats dibutyryl cyclic AMP provoked an immediate dramatic increase in phosphoenolpyruvate carboxykinase activity above the starvation level even if mRNA synthesis was inhibited by cordycepin. However, cyclic AMP was ineffective in increasing enzyme activity in livers of adrenalectomized rats. From these results it is suggested (i) that in starved rats the adaptation to the enhanced glucose demand provoked by cold exposure includes the induction of hepatic phosphoenolpyruvate carboxykinase above the starvation level, (ii) that this induction is due to the cold-induced increase in hepatic cyclic AMP levels, (iii) that cyclic AMP stimulates enzyme synthesis at a post-transcriptional step and (iv) that the cold-induced cyclic AMP-mediated induction of phosphoenolpyruvate carboxykinase above the starvation level requires the "permissive" effect of glucocorticoids.

Kinetics and regulation of synthesis of the major polypeptides of thylakoid membranes in Chlamydomonas reinhardtii y-1 at elevated temperatures

Etiolated cells of Chlamydomonas reinhardtii y-1 exhibit rapid and linear initial kinetics of greening when exposed to light at 38 degrees C. The initial rate of chlorophyll accumulation under these conditions is greater than the maximal rate during greening at 25 degrees C. Synthesis of the major polypeptides of thylakoid membranes within intact cells was assayed during greening by the incorporation of [3H]leucine and the subsequent electrophoresis of total cellular protein on polyacrylamide gels in the presence of sodium dodecyl sulfate. At 38 degrees C the major membrane polypeptides (about 28,000 and 24,000 daltons in mass) were synthesized at a linear rate after exposure of the cells to light, with no evidence of a lag period. A 1-2 h preincubation in the dark at the higher temperature was necessary to achieve linear initial kinetics. Actinomycin D inhibited synthesis of the membrane polypeptides if added at the beginning of a 2 h dark preincubation, but not when added near the end. These results suggested that transcription of the messenger RNA for the membrane polypeptides occurred during the dark period at 38 degrees C. But the major membrane polypeptides were not made by y-1 cells in the dark. The wavelengths of light most effective in eliciting production of the membrane polypeptides were the same as those allowing chlorophyll synthesis. In contrast, wild type cells, which are capable of chlorophyll synthesis in the dark, also make the membrane polypeptides in the dark. The data indicate that at elevated temperatures synthesis of the major thylakoid membrane polypeptides is controlled at a posttranscriptional step, and that this reaction normally proceeds only under conditions which permit reduction of protochlorophyllide.

Control of bacteriophage Lambda repressor synthesis: Regulation of the maintenance pathway by the cro and cI products

Activation of p rm, the promoter at which cI transcription is initiated in immune bacteriophage lambda lysogens, requires no phage genes or sites outside the cI region, cro product, however, is able to reduce fivefold the efficiency of cI expression from this promoter. cro product does not inactivate λ repressor, the cI gene product, and cro product also acts effectively in the presence of active repressor. Therefore, cro product seems to block directly the promoter p rm at a site which can not be protected by repressor binding to the right operator O r. Since cro product remains an effective inhibitor of p rm in a bacterial strain in which it can not block efficiently leftward transcription of the N-cIII operon or rightward transcription of the cro-cII operon, cro product probably has three separate target sites; one near p 1 at which it regulates N and cIII, one near p r at which it regulates cro and cII, and a third near p rm at which it blocks cI. My experiments indicate that repressor must be bound to the right operator O r to activate either the promoter p rm or a post-transcriptional step in cI polypeptide synthesis. In the absence of cro product, cI expression from p rm is not constitutive. Pre-existing repressor stimulates sevenfold the initial rate of active repressor synthesis by λ NcrocII. After 20 generations growth at high temperatures, only 3 to 10% the normal repressor antigen level is seen in defective, cro-minus lysogens containing any of four different cI ts alleles. Studies on the cI ts857 antigen, presented in the Appendix, show that these low antigen levels cannot simply be a consequence of poor antigen formation and stability. Instead they must reflect at least a fourfold decrease in the rate of cI polypeptide synthesis. The weakly constitutive right operator mutation v 3 reduces the maximum temperature at which cI ts857 repressor synthesis can be maintained in cro-minus lysogens. cro + phage bearing more constitutive right operator mutations block repressor synthesis in trans by making cro product. These operator mutations are much more effective at blocking repressor synthesis in cis, however, which also argues that repressor must be bound to the operator if there is to be efficient cI polypeptide synthesis initiated at the promoter p rm.

Synthesis and turnover of nuclear and cytoplasmic polyadenylic acid in mouse L cells

A study was made of the kinetics of adenosine incorporation into the poly(A) segments of heterogeneous nuclear RNA, polyribosomal messenger RNA and total cytoplasmic RNA of mouse L cells. Special care was taken to ensure that the precursor pools used for incorporation remained at a constant specific activity over the entire course of an experiment. A comparison of the labeling of the poly(A) and the non-poly(A) portions of mRNA indicated that polyadenylation is a relatively late post-transcriptional step, occurring in the latter third of the interval between transcription of the mRNA precursor and appearance of the mRNA in cytoplasmic polyribosomes. The rates of labeling of messenger poly(A) and total cytoplasmic poly(A) reach a maximum after only a very brief lag and do not increase thereafter, in spite of that fact that the specific activity of nuclear poly(A) continues to increase for several hours. Such a result is not compatible with a simple precursor-product relation between all of the nuclear and all of the cytoplasmic poly(A), but rather, it suggests that there is a reasonably large intranuclear turnover of poly (A). The fact that nuclear poly(A) is not quantitatively converted to cytoplasmic poly(A) indicates that polyadenylation of a heterogeneous RNA molecule is not sufficient to ensure that it will be properly processed and transported to the cytoplasm.

Mechanisms of hormonal induction of enzymes

The study of enzyme induction by hormones has provided an important experimental model for the investigation of both the molecular mechanisms of hormone action as well as the regulation of specific gene expression in higher organisms. The exact mechanisms by which hormones induce enzymes are as yet unknown. The ability to study enzyme induction in relatively simple experimental systems such as cells in tissue culture allows the investigator to assess unequivocally the role of single hormones and to pose mechanistic questions. It is hoped that advances in genetic approaches to enzyme induction will accelerate progress in understanding these mechanisms. Studies of tyrosine aminotransferase induction by glucocorticoids in the HTC cell system have led to a model of enzyme induction in which the inducing steroids are thought to antagonize the action of a posttranscriptional repressor. The emphasis of this model, in contrast to those based on bacterial models, is that regulation of specific gene expression need not operate at the genetic or transcriptional level; i.e., by stimulating specific mRNA synthesis. Transcripitonal controls surely exist in animal cells, but their role in specific enzyme induction remains to be established. In addition to glucocorticoids, several other hormonal factors including insulin, a macromolecular factor in serum, and dibutyryl cyclic-AMP, can also enhance TAT synthesis, all probably affecting separate and distinct posttranscriptional steps in the synthesis of a single protein.

Effects of protein synthesis inhibitors on the induction of a labile chicken plasma coagulation factor by preparations of phytoene and substituting factors

The phytoene-dependent component of the labile coagulation factor activity complex in chicken plasma (the π-factor) responds uniformly to intravenous administration of preparations of phytoene, and of three unidentified “phytoene-substituting” factors: one occuring in lard, and two others in hog liver fat. In all cases there was an initial lag of about 10 min, followed by a rapid increase in π-factor concentration, reaching the maximum level about 35 min after the injection. The configuration of “active phytoene” and the structures of the phytoene-replacing factors remain unknown. The π-factor response was blocked and arrested by simultaneous and delayed injections, respectively, of cycloheximide and puromycin, whereas simultaneous or prior injection of actinomycin D had no effect on the response to the dietary factors. The data suggest that a post-transcriptional step in π-factor biosynthesis is regulated by the dietary factors or a common metabolite. Control of a specific pre-translational step and of an early step in reactions governing a specific post-translational process are considered as possible modes of action.

Evidence for translational regulation of specific enzyme synthesis by N 6, O 2′-dibutyryl cyclic AMP in hepatoma cell cultures

N 6,O 2′-dibutyryl cyclic 3′:5′ AMP (dibutyryl cAMP) produces a rapid increase in the activity of phosphoenolpyruvate carboxykinase (PEPCK) in Reuber H35 hepatoma cell cultures. This effect is shown to result from a stimulation of enzyme synthesis. Low concentrations of actinomycin D completely block the response of PEPCK to dexamethasone but not that at early intervals after addition of dibutyryl cAMP. These results suggest that dibutyryl cAMP affects a post-transcriptional step in PEPCK synthesis. Superinduction of tyrosine transaminase after addition of high concentrations of actinomycin D is observed with both dexamethasone and dibutyryl cAMP; PEPCK does not exhibit this phenomenon with either inducer.

Regulation of Tyrosine α-Ketoglutarate Transaminase in Rat Liver: IX. STUDIES OF THE MECHANISMS OF HORMONAL INDUCTIONS IN CULTURED HEPATOMA CELLS

Abstract Synthesis of the messenger RNA that codes for tyrosine α-ketoglutarate transaminase (l-tyrosine:2-oxo-glutarate aminotransferase, EC 2.6.1.5) was blocked by actinomycin and its stability was determined in cultured hepatoma cells by measuring rates of transaminase synthesis. By this criterion the transaminase messenger RNA is degraded with a half-life of 2 to 3 hours in both H-35 and HTC cell lines; this rate is not changed by hydrocortisone. Transaminase synthesis is decelerated by high and low concentrations of actinomycin; high concentrations of this antibiotic block transaminase degradation as well. At low concentrations of actinomycin, transaminase induction by hydrocortisone is blocked but induction by insulin is unaffected for a period of time consistent with messenger stability. Preliminary treatment with hydrocortisone primes the cells to respond to insulin with a rapid increase in enzyme, but preliminary treatment with insulin does not alter the response to hydrocortisone. With the use of experimentally derived rates of degradation of both the transaminase and its messenger RNA, a mathematical model was developed which satisfactorily predicts kinetic behavior of the enzyme level upon addition and withdrawal of inducing hormones. The data are consistent with the conclusion that hydrocortisone acts transcriptionally while insulin acts on some posttranscriptional or translational step in enzyme synthesis. We find no indication of posttranscriptional mechanisms in the steroid-mediated induction. A discussion of the mathematical model used is appended.

Ribosome formation in rat liver: Evidence for post-transcriptional control

An investigation of the amount of label incorporated from [6- 14C]orotic acid into the nuclear and cytoplasmic ribosomal RNA of non-proliferating rat liver, during a 1-h treatment with several inhibitors of protein biosynthesis, gave the following results: 1. 1. The incorporation into nuclear 45-S, 28-S and 18-S RNA of liver was increased during a 1-h treatment with anisomycin and cycloheximide by 50%, and with puromycin by 25%; 8-azaguanine showed little effect. 2. 2. The appearance of label in newly synthesized ribosomes increased approx. 25% after cycloheximide or anisomycin treatment; in contrast, this parameter was reduced to 25% that of the controls after puromycin treatment, and to 50% that of the controls after 8-azaguanine treatment. This differential effect of the inhibitors on ribosome transport was also observed in the slow growing Hepatoma 7800. 3. 3. The size of the ribosomal subunit (60 S and 40 S) pools were not significantly altered by the inhibitors, even though puromycin and 8-azaguanine caused extensive conversion of the polyribosomes to monomers and dimers. The results suggest that the differential effect of the inhibitors on ribosome formation and polyribosome structure are related. They further suggest that this feed-back control, which may involve monomeric ribosomes, acts at a post-transcriptional step. There appears to be a pool of ribosomal proteins in non-proliferating rat liver and in the slow growing hepatoma which is sufficient to support ribosome synthesis for at least 1 h.