Kinetics Of Protein Synthesis Research Articles

A positive regulatory gene is required for accumulation of the functional messenger RNA for the glucose-repressible alcohol dehydrogenase from Saccharomyces cerevisiae

The amount of glucose-repressible alcohol dehydrogenase is regulated by the amount of its functional messenger RNA. ADHII † † Abbreviations used: ADHII, alcohol dehydrogenase, isoenzyme II: ADHI, alcohol dehydrogenase, isoerizyme 1: SDS, sodium dodecyl sulfate. protein was detected by a radioimmune assay and differentiated from ADHI, the classical ADH isozyme, by limited proteolysis with Staphylococcus aureus protease. When yeast containing the wild-type alleles for ADR2 (the ADH II structural locus) and for ADR1 (its positive regulatory gene) were pulse-labeled with [ 35S]methionine during derepression, radioactive label accumulated in the antibody-precipitated ADHII coterminously with the appearance of ADHII activity. The kinetics of functional ADHII mRNA appearance during derepression in this strain were shown to be the same as those for ADHII protein synthesis in vivo when RNA, extracted from derepressed cells, was translated in a wheat germ cell-free translation system. The role of the positive regulatory gene, ADR1, in ADHII expression was analyzed using two strains mutated at that locus. Yeast containing the adr1-1 allele are incapable of derepressing ADHII activity. When this strain was pulselabeled with [ 35S]methionine during derepression, approximately one-tenth to one-twentieth the level of ADHII protein synthesis was detected as in the wild-type strain. When RNA was extracted during derepression from cells containing the udr1-1 allele and translated in a wheat germ cell-free system, little functional ADHII mRNA was found to be present. The role of the ADR1 gene was further analyzed using a strain containing the ADR1-5 c allele, which allows constitutive synthesis of ADHII activity. In this strain during glucose repression. ADHII protein synthesis and amount of functional mRNA were at levels comparable to those found for the wild-type strain after complete derepression. Similar kinetics of ADHII protein synthesis and of mRNA accumulation during derepression were observed in the strain carrying the ADR1-5 c allele when compared to that carrying the ADR1 allele, but the absolute amounts were greater by three- to fourfold in cells containing the ADR1-5 c allele. These results indicate that the ADR1 gene acts to increase the level of functional ADHII mRNA during derepression.

A dominant lethal temperature-sensitive mutant of herpes simplex virus type 1

A temperature-sensitive ( ts) mutant of herpes simplex virus type 1 (HSV-1), ts P23, markedly inhibited the growth of the wild-type virus from which it was derived in mixed infectiobs at the nonpermissive temperature. When cells were mixedly infected with equal multiplicities of ts P23 and other HSV-1 mutants, complementation was inefficient. However, complementation indices increased as the multiplicity of ts P23 decreased relative to that of the other mutant, indicating that interference by tsP23 is a function of the dose of the mutant gene. Biochemically, ts P23 is DNA-negative, overproduces certain immediate early and delayed early viral polypeptides, and underproduces late, structural gene products. Patterns of viral DNA and protein synthesis in cells mixedly infected with ts P23 and the wild-type virus at the nonpermissive temperature were similar to those of cells infected with the mutant alone at this temperature. Temperature-shift studies and the kinetics of interference by ts P23 with the growth of the wild-type virus indicate that the mutant is arrested early in the replicative cycle. In support of this hypothesis is the observation that viral polypeptide synthesis in cells infected with ts P23 at 39° was similar but not identical to that observed in cells infected with the wild-type virus in the presence of phosphonoacetic acid, a drug which is thought to act directly on the viral DNA polymerase molecule, a delayed-early viral gene product. The interfering properties of the mutant, the timing of the inhibitory effect, and the kinetics of protein synthesis in mutant-infected cells at 39° indicate that ts P23 is a dominant lethal mutant with a primary defect in a “trans” acting, delayed early gene function essential for viral DNA synthesis.

Preparation and partial characterization of cell-free protein-synthesizing extracts from Tetrahymena pyriformis.

1. We have examined methods necessary for preparing post-mitochondrial supernatants from Tetrahymena pyriformis strain HSM that are capable of efficient cell-free protein synthesis. 2. The requirements for optimum synthesis in these extracts are described. 3. Data relating to the kinetics of protein synthesis and the initiation capacity of these supernatants are presented.

Effects of cyclic AMP on the kinetics of serum protein synthesis in cultured mouse hepatoma cells

The mouse hepatoma cell line, Hepa, was cultured in the presence of either 1 mM N 6, O 2′ -dibutyryl cyclic AMP (Bt 2cAMP), 0.5 mM 3-isobutyl-1-methylxanthine, or 1 μg/ml cholera toxin. The synthesis and secretion of albumin, α-fetoprotein, and transferrin were elevated above controls by 24 h reaching two- to fourfold stimulations within 72 h. These effects were reversible and were specific for the serum proteins. The stimulation of serum protein synthesis was accompanied by a decrease in the rate of cell proliferation. Protein synthetic parameters were analyzed in Hepa cells 72 h after exposure to N 6,O 2′ -dibutyryl cyclic AMP. The cellular rate of albumin synthesis was increased fourfold and the relative rate of albumin synthesis was increased approximately threefold. N 6,O 2′ -Dibutyryl cyclic AMP did not affect the size distribution of either total Hepa polyribosomes or of albumin-synthesizing polyribosomes. The elongation rate on total mRNA and on albumin mRNA was decreased by approximately 40%. These results indicate that the rate of initiation of total Hepa mRNA and of albumin mRNA also decreased by 40%. The nonspecific nature of the N 6,O 2′ -dibutyryl cyclic AMP effect on Hepa protein synthetic parameters must be due to an alteration in the level of a common substrate, perhaps ATP. The specific threefold increase in the relative rate of albumin synthesis with no alteration in polyribosome sizes requires a threefold increase in the relative amount of functional albumin mRNA in Hepa cells. This prediction was confirmed by cell-free translation of Hepa polyribosomes.

Varying patterns of protein synthesis in Drosophila during heat shock: Implications for regulation

The heat-shock response of Drosophila involves the vigorous induction of a small number of new messenger RNAs and proteins as well as the repression of most preexisting RNA and protein synthesis. The experiments presented here characterize the kinetics of messenger RNA and protein synthesis at different temperatures and the patterns of induction under a variety of culture conditions. In addition to providing practical information for further studies of the heat-shock response, the data provide some valuable insights into the nature of the response. In particular, the patterns of induction and repression are not simple functions of the degree of temperature elevation, but vary strikingly in different media and depend strongly upon the speed of the temperature increase. Several heat-shock proteins are shown to have very individual induction characteristics with respect to the temperatures at which they are maximally induced, the range of temperatures over which they are synthesized, and the kinetics of their induction. Thus, although this system has often been viewed as a simple, coordinate induction, it now appears that the various heat-shock genes can be, to a rather considerable extent, regulated independently of one another. The evidence further suggests that the patterns of protein synthesis in heat-shocked cells are regulated by mechanisms which act at several different levels of gene expression.

Protein synthesis kinetics with ribosomes from temperature-sensitive mutants of Escherichia coli

The kinetics of MS2 ribonucleic acid (RNA) directed protein synthesis have been investigated at seven temperatures between 30 and 47 degrees C by using ribosomes isolated from a wild type strain and seven temperature-sensitive mutants of Escherichia coli. The amount of MS2 coat protein formed at each temperature was determined by gel electrophoresis of the products formed with control ribosomes. With ribosomes from each of the mutant strains, the activation energy required to drive protein synthesis below the maximum temperature (up to 40 degrees C) was increased relative to the control (wild type) activity. Preincubation of the ribosomes at 44 degrees C revealed the kinetics of thermal inactivation, with ribosomes from each of the mutants having a half-life for inactivation less than that of the control ribosomes. A good correlation was observed between the relative activity of the different ribosomes at 44 degrees C and their relative rate of thermal inactivation. Mixing assays allowed the identification of a temperature-sensitive ribosomal subunit for each of the mutants. Defects in one or more of three specific steps in protein synthesis (messenger RNA binding, transfer RNA binding, transfer RNA binding, and subunit reassociation) were identified for the ribosomes from each mutant. The relationship between temperature sensitivity and protein synthesis in these strains is discussed.

The analysis of a temperature-sensitive (ts) mutation influencing the expression of heat shock-inducible genes in Drosophila melanogaster.

The effect of a ts-mutation belonging to the cell-lethal type on protein and RNA synthesis under heat shock conditions has been investigated. The mutation studied, l(l)ts-403, localized in the X-chromosome has significant effect on the kinetics of protein synthesis in salivary gland subjected to heat treatment under in vivo and in vitro conditions. The animals homozygous for l(l)ts-403 are characterized by a rapid drop of label incorporation into heat shock proteins. It is shown that the mutation affects not only the kinetics of heat-shock proteins synthesis but their electrophoretic pattern as well. The experiments performed enable us to conclude that the presumptive regulatory gene realized its action at the level of transcription of heat shock loci.

Involvement of DNA gyrase in replication and transcription of bacteriophage T7 DNA

Growth of bacteriophage T7 is inhibited by the antibiotic coumermycin A1, an inhibitor of the Escherichia coli DNA gyrase. Since growth of the phage is insensitive to the antibiotic in strains containing a coumermycin-resistant DNA gyrase, this enzyme appears to be required for phage growth. We have investigated the effect of coumermycin on the kinetics of DNA, RNA, and protein synthesis during T7 infection. DNA synthesis is completely inhibited by the antibiotic. In addition, coumermycin significantly inhibits transcription of late but not early genes. Thus, E. coli DNA gyrase may play an important role in transcription as well as in replication of T7 DNA.

Interaction between membrane properties and protein synthesis: In vitro synthesis of membrane proteins during erythropoiesis

Summary Membrane protein synthesis was investigated by incubating rabbit reticulocytes, in vitro, with radioactive amino acids. The kinetics of membrane protein synthesis showed linear incorporation for approx. 15 min, after which there was only a slight increase in incorporation. On the other hand, intracellular protein synthesis was linear for an incubation period of 60 min. Membranes isolated from such rabbit reticulocytes were analysed on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Two major radioactive bands were found in the 50–60 000 D region, whilst another labelled band had a molecular weight of 43 000 D. This latter band had an electrophoretic mobility identical with rabbit muscle actin (and chick brain actin), when run on one-dimensional SDS polyacrylamide gels. Absolute identity between rabbit brain actin and a newly synthesized reticulocyte membrane protein was shown by comigration on a two-dimensional (first dimension isoelectric focusing and second dimension SDS gel) electrophoresis system. Another band that was radioactively labelled was found to have a molecular weight of approx. 32 000 D. Separation of reticulocytes into different age groups showed that young reticulocytes synthesized a membrane protein species that was not radioactively labelled in the old reticulocyte population.

An investigation of the stability of messenger RNAs in cell-free, translational systems from uninfected and mengovirus-infected Ehrlich ascites tumor cells

The stabilities and translation of Ehrlich ascites tumor cell poly(A)-containing mRNA and mengovirus RNA in fractionated cell-free protein synthesizing systems from uninfected and mengovirus-infected Ehrlich ascites tumor cells were studied. During incubation of the systems about 20% of the input RNA is reduced in size and associated with ribosomes engaged in polypeptide synthesis; the remainder is rapidly degraded by RNases. At the end of active translation, both mRNA and nascent proteins are bound to polysomes which are of the same size as those formed during active protein synthesis. The kinetics of protein synthesis closely follow those of RNA hydrolysis. The stabilities of mengovirus RNA and poly(A)-containing mRNA from Ehrlich ascites tumor cells are the same in both systems.

Non-histone protein synthesis during G1 phase and its relation to DNA replication.

The kinetics of non-histone chromosomal protein (NHCP) synthesis were studied in Chinese hamster ovary (CHO) plateau phase cells stimulated to proliferate and were compared to NHCP synthesis kinetics in two populations of synchronous G1 traversing cells. In all cases, NHCP synthesis rates increase 3- to 5-fold as cells traversed G1 and attained maximum values one hour before semi-conservative DNA replication began. Similar to results in synchronous G1 cells, the molecular weight distributions of the NHCP fraction from stimulated plateau phase cells underwent only minor changes, measured by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis, as these cells moved toward S phase. Yet, during this progression after plateau phase and in the transition from early G1 to late G1 in synchronous cells, the total NHCP fraction increased significantly (1.5-2-fold) in amount per cell. These data indicate that plateau phase cells are similar to early G1 cells both in terms of their amounts of non-histone per cell and in their subsequent NHCP synthesis kinetics as they move toward S phase. These results extend previous findings which suggested that NHCP synthesis was coupled to DNA replication and demonstrate that the increased NHCP synthesis and accumulation in chromatin may be a biochemical marker for G1 progression.

Synthesis of deoxyribonucleic acid, ribonucleic acid, and protein during sporulation of Clostridium perfringens

The kinetics of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein synthesis as well as protein breakdown during sporulation by Clostridium perfringens were determined. Maximum levels of DNA and net RNA synthesis occurred 3 and 2 h, respectively, after inoculation of sporulation medium. The rate of RNA synthesis decreased as sporulation progressed. Deoxyadenosine increased uptake of [14C]uracil and [14C]thymine but depressed the level of sporulation and the formation of heat-resistant spores when added at concentrations above 100 mug/ml. Unlike Bacillus species, net protein synthesis, which was sensitive to chloramphenicol inhibition, continued during sporulation. The rate of protein breakdown during vegetative growth was 1%/h. During sporulation this rate increased to 4.7%/h. When added to sporulation medium at 0 time chloramphenicol reduced protein breakdown to 1%/h. If added at 3 h the rate decreased to 2.1%/h. The role of proteases in this process is discussed.

Kinetics of protein synthesis and release in the STH-cell of the mouse anterior pituitary as revealed by electron microscopic autoradiography of 3H-amino acids.

In order to investigate the kinetics of the synthesis and release of the secretory protein in the STH-secreting cell of the pituitary anterior lobe, studies were made using the electron microscopic autoradiography of 3H-amino acids. Each mouse received an intravenous injection of 2.8 mCi of 3H-amino acids consisting of equal amounts of 3H-leucine, 3H-tyrosine, 3H-lysine, 3H-histidine and 3H-glycine. The animal was sacrificed at 3, 5, 15 and 30 min, 1, 2, 4, 8 and 24 hrs after the injection. Although the whole silver grains are very few 3 min after the injection, half of the grains are recognized over the r-ER (47.1%); the Golgi apparatus already shows a few grains (5.9%). Total silver grains over the STH-cell increase in number with the lapse of time; the ratio of the grain number over the r-ER to the total grains over the whole cell decreases gradually, while the ratio of the grain number over the Golgi apparatus increases to reach a peak about 30 min after the injection (40.1%) and then decreases gradually. The ratio of the number of labelled granules to that of total granules increases slowly until 24 hrs after the injection (3 min: 0%, 5 min: 0.8%, 30 min: 3.9%, 2 hrs: 5.0%, 8 hrs: 8.0%, 24 hrs: 12.3%). It is concluded that the turnover in synthesis and release of exportable protein in the mouse STH-cell is very slow as compared with that of the thyroid and parathyroid cell.

Control of Haemoglobin Synthesis

A detailed examination of the kinetics of protein synthesis in rabbit reticulocytes in the presence of the iron chelating agent 2,2'-dipyridyl showed that between 30 degrees C and 42 degrees C there were characteristically two distinct phases of protein synthesis. An initial phase (I), in which no inhibition of protein synthesis was apparent, was followed by a gradual decline in the rate of protein synthesis leading to the second phase (II) in which protein synthesis occurred at a linear but inhibited rate for extended periods. In contrast, below 30 degrees C, incubation in the presence of dipyridyl caused no inhibition of protein synthesis. Between 30 degrees C and 42 degrees C the duration and amount of protein synthesis occurring in phase I before the onset of inhibition were inversely related of the inhibition as was the final rate of incorporation in phase II. During phase II, a partial reversal of the inhibition caused by dipyridyl was obtained by lowering the incubation temperature. This resulted in a burst of protein synthesis at the uninhibited rate until the amount of protein synthesis reached the same level as that in reticulocytes maintained continuously with dipyridyl at the lower incubation temperature. This burst of synthesis was observed in reticulocytes which had been held in phase II for as long as 90 min. It was also possible to reverse the inhibition by addition of haemin to cells in phase II. At any particular incubation temperature, a fixed number of rounds of protein synthesis had to occur before the onset of phase II became apparent. By the use of puromycin we showed that this was not a requirement for the synthesis of globin or of any other protein. We believe that this critical amount of protein synthesis reflects the residual ability of reticulocytes to initiate new protein chains in the absence of concurrent haem synthesis. Reticulocytes preincubated in the presence of cobaltous ions showed almost no inhibition of protein synthesis upon subsequent incubation with dipyridyl. The results are compared to those obtained in reticulocyte lysates and are discussed in terms of current theories to account for control of protein chain initiation by haemin.

Radioactive labelling and characterization of the products of activated mouse lymphocytes.

For chemical characterization of the products of activated lymphocytes a radioactive double-label technique was developed which allows one to distinguish those products synthesized either de novo or in increased amounts by the stimulated culture. Spleen cells from Balb/c mice were cultured in serum-free medium in the presence or absence of concanavalin A and simultaneously labelled with radioactive leucine. Optimal culture conditions were established by determining parameters such as cell density, mitogen concentration, and kinetics of protein synthesis following stimulation. Combined supernatants of stimulated and unstimulated cultures each labelled with either [3H]leucine or [14C]leucine were fractionated on Sephadex G-75. Materials derived from control or stimulated supernatants both yielded a qualitatively similar radiolabelled profile. The isotope ratio of stimulated to nonstimulated culture, however, showed a broad peak at KD 0--.35 (approx. mol. wt 75000-20000) which was further analyzed by isoelectric focusing. Pools of every two fractions were focused in polyacrylamide gels at pH 3.5-10. By determining the isotope ratio, the isoelectric point, and the KD (mol wt), it was possible to distinguish at least 24 molecules which had been produced only, or in greater degree, by the stimulated culture.

Protein synthesis in stimulated lymphocytes: II. Amino acid requirements

The amino acids required for protein synthesis by phytohemagglutinin (PHA) and antigen stimulated lymphocytes have been determined by measuring the kinetics of protein synthesis in amino acid deficient media. Stimulated lymphocytes have a partial requirement for serine in addition to the 13 amino acids which Eagle (1) found to be essential for mammalian cell cultures. Asparagine is not required for the lymphocyte response. This observation supports the suggestion that l-asparaginase inhibits lymphocyte blastogenesis by its action on the membrane rather than by depletion of exogenous asparagine. With the exception of serine, tryptophan, and isoleucine, the omission of an essential amino acid results in complete inhibition of PHA induced protein synthesis. In the absence of either tryptophan or isoleucine, the rate of protein synthesis is unaffected during the first 30 hr of incubation but subsequently declines rapidly to that of unstimulated cultures, suggesting a possible role for tryptophan and isoleucine in the regulation of the lymphocyte cell cycle. The inhibition of protein synthesis by amino acid deficiencies is reversible upon replacement of the missing amino acid.

On the kinetics of poliovirus-specific protein and RNA synthesis in poliovirus-infected cells.

On the kinetics of poliovirus-specific protein and RNA synthesis in poliovirus-infected cells.

Effects of temperature-induced phase changes in membranes on protein synthesis by bound ribosomes

The effect of temperature on the kinetics of protein synthesis by both free and membrane-bound ribosomes has been investigated. Free ribosomes isolated from rat liver conform to the predictions of the Arrhenius equation in that a plot of log of the amino acid incorporation rate against the reciprocal of the absolute temperature gives a simple linear relationship from which an Arrhenius activation energy( E) of 25.5 kcal/mole is calculated. However, similar plots of data for the protein synthetic activity of rough endoplasmic reticulum show two linear regions giving Arrhenius activation energies of 16 kcal/mole above 22 °C and 43 kcal/mole below this temperature. A phase change in the lipids of rough endoplasmic reticulum was demonstrated at 22 °C by electron spin resonance spectroscopy using a suitable lipophilic spin label. The importance of the membrane in the origin of the discontinuity in the Arrhenius plots was confirmed by experiments with reconstituted rough endoplasmic reticulum derived from inactive “stripped” membranes and free ribosomes. We conclude that phase changes occurring in the membrane as a consequence of lipid reorganization can influence the activity of ribosomes associated with the membranes.

The synthesis of acidic chromosomal proteins during the cell cycle of HeLa S-3 cells. II. The kinetics of residual protein synthesis and transport.

The kinetics of acidic residual chromosomal protein synthesis and transport were studied throughout the cell cycle in HeLa S-3 cells synchronized by 2 mM thymidine block and selective detachment of mitotic cells. Pulse labeling the cells with leucine-(3)H for 2 min and then "chasing" the radioactive proteins for up to 3 hr showed that the amount of protein synthesized, transported, and retained in the acidic residual chromosomal protein fraction is greater immediately after mitosis and later in G(1) than in the S or G(2) phases of the cell cycle. During S, only 20-25% of the proteins synthesized and transported to the acidic residual chromosomal protein fraction are chased during the first 2 hr after pulse labeling, whereas up to 40% of the material entering the residual nuclear fraction in mitosis, G(1), and G(2) leaves during a 2 hr chase. Polyacrylamide gel electrophoretic profiles of these proteins, at various times after pulse labeling, reveal that the turnover of individual polypeptides within this fraction has kinetics of synthesis and turnover which are markedly different from one another and undergo stage-specific changes.

Division cycle of Myxococcus xanthus. 3. Kinetics of cell growth and protein synthesis.

The kinetics of cell growth and protein synthesis during the division cycle of Myxococcus xanthus was determined. The distribution of cell size for both septated and nonseptated bacteria was obtained by direct measurement of the lengths of 8,000 cells. The Collins-Richmond equation was modified to consider bacterial growth in two phases: growth and division. From the derived equation, the growth rate of individual cells was computed as a function of size. Nondividing cells (growth phase) comprised 91% of the population and took up 87% of the time of the division cycle. The absolute and specific growth rates of nondividing cells were observed to increase continually throughout the growth phase; the growth rate of dividing cells could not be determined accurately by this technique because of changes in the geometry of cells between the time of septation and physical separation. The rate of protein synthesis during the division cycle was measured by pulselabeling an exponential-phase culture with radio-active valine or arginine and then preparing the cells for quantitative autoradiography. By measuring the size of individual cells as well as the number of grains, the rate of protein synthesis as a function of cell size was obtained. Nondividing cells showed an increase in both the absolute and specific rates of protein synthesis throughout the growth phase; the specific rate of protein synthesis for dividing cells was low when compared to growthphase cells. Cell growth and protein synthesis are compared to the previously reported kinetics of deoxyribonucleic acid and ribonucleic acid synthesis during the division cycle.