Overall Rate Of Protein Synthesis Research Articles

Protein metabolism in SV40-infected cells

Simian virus 40-induced alterations in protein metabolism were investigated in confluent monolayers of BS-C-1 monkey kidney cells. Viral-induced changes were observed which were specific for both the prereplicative and the replicative phases of infection. The prereplicative phase is that period of the infection prior to the onset of viral DNA synthesis, which begins at 19–20 hr. A viral-induced stimulation of protein synthesis is first observable at about 10–12 hr, and by 20 hr after infection the rate of protein synthesis has increased to more than 20% above that of mock-infected cells. Gel electrophoresis of the proteins synthesized during the prereplicative period did not reveal any differences from control cells in any cell fraction except the cytosol, i.e., the nonparticulate portion of the cytoplasm. In this fraction several new species were observed, but together they did not constitute more than 1% of the overall rate of protein synthesis. An inhibition of labeling of one, and possibly two, host proteins was also discerned in the cytosol. Each of the polypeptide changes just described was also observed in infected cells which had been treated with cytosine arabinoside. This confirms the assignment of these effects to the early phase of infection, as it has been well-established that cytosine arabinoside blocks viral DNA synthesis and all late viral functions, but has little effect on early events. During the replicative period the rate of protein synthesis continues to increase to more than 50% above that of mock-infected cells. Starting at 20 hr postinfection, proteins destined for the nucleus are synthesized at higher relative rates than those of cytoplasmic fractions. As a consequence, a very large progressive accumulation of protein in the nucleus begins at about 40 hr after infection. Much of this increased nuclear mass could be accounted for as progeny virus particles found in the membranous fraction of sonicated nuclei. Gel electrophoresis of newly-made proteins late in infection showed that the two major viral structural proteins constituted about 20% of cellular protein synthesis. There was an additional viral-induced nonstructural polypeptide in the cytosol which constituted less than 1% of the newly-made proteins. As expected, these three new proteins were not synthesized in infected cells which had been treated with cytosine arabinoside. It was also observed that the labeling of the major host protein in the nuclear membrane was inhibited during the late phase of infection.

Intracellular amino acids and protein synthesis in Hela cells

(1) When the intracellular amino acid pool is prelabelled and subsequently chased in non-radioactive medium, the radioactivity of the amino acid pool is not found to have been incorporated into protein. (2) Leucine transport into Hela cells is reduced in the presence of 10 mM valine in the medium. This results in a lower specific radioactivity of leucine in the intracellular amino acid pool. However, neither the overall rate of protein synthesis nor the incorporation of radioactive leucine into protein is affected. From these experiments it is concluded that incoming amino acids entering the intracellular amino acid pool are not used for de novo synthesis of protein.

Polyamine synthesis during lymphocyte activation: Induction of ornithine decarboxylase and S-adenosyl methionine decarboxylase

Lymphocyte stimulation by phytohaemagglutinin (PHA) is accompanied by marked increases in the activities of ornithine decarboxylase and S-adenosyl methionine decarboxylase, two key enzymes for the synthesis of polyamines. Both enzymes increase in a biphasic manner, with the rises in S-adenosyl methionine decarboxylase preceding the increases in ornithine decarboxylase. The initial rises precede the initiation of DNA synthesis, and seem to correlate with the increased rate of ribosomal RNA synthesis. Selective inhibition of ribosomal RNA synthesis inhibits the increases in the activity of both enzymes, especially ornithine decarboxylase, more than the increase in the overall rate of protein synthesis. Both enzymes are metabolically unstable and have half-lives of less than 1 h, although the half-life of ornithine decarboxylase depends on the amino acid concentration in the culture medium. While effects of PHA on the stability of the enzymes have not been ruled out, at least part of the PHA-dependent increases in activity are due to increased synthesis or activation of the enzymes. The synthesis of S-adenosyl-methionine decarboxylase declines rapidly after inhibition of RNA synthesis, but ornithine decarboxylase activity declines at about the same rate as protein synthesis as a whole. The activities of both enzymes also increase during lymphocyte stimulation by concanavalin A, lentil extract and staphylococcal filtrate.

Efficiency of protein and messenger RNA synthesis in bacteriophage T4-infected cells of Escherichia coli

Several basic parameters related to the synthesis of protein and RNA in cells of Escherichia coli B infected with bacteriophage T4 have been measured. (a) The over-all rate of protein synthesis, estimated by incorporation of four different amino acids, decreases to 42% shortly after infection. (b) A general method was developed for measuring the average peptide chain growth-rate under steady-state conditions. It involves separation on sodium dodecyl sulfate—acrylamide gels of total protein labeled for different short periods of time, followed by an analysis of the labeling kinetics of proteins in different molecular weight classes. By this method the peptide chain growthrate in T4-infected cells was found to be 5.6 amino acids per second at 30 °C compared to 11.4 amino acids per second measured in exponentially growing cells under the same growth conditions. (c) The proportion of ribosomes in polysomes before and at different times after T4 infection was determined by sucrose gradient analysis. No significant differences were observed. (d) The stimulating activity of RNA extracts in an in vitro protein-synthesizing system was measured and used as an estimate of messenger RNA. By this criterion the level of mRNA is at least as high and probably higher after infection. (e) Residual protein synthesis was followed after addition of rifampicin, an antibiotic that blocks initiation of RNA synthesis, and under these conditions the protein-synthesizing capacity decreased with a half-life of 2.2 minutes in uninfected, and 4.0 minutes in T4-infected cells (at 30 °C). The decrease in over-all rate of protein synthesis observed after infection is thus accounted for by the decrease in peptide chain growth-rate. The twofold decrease in peptide chain growth-rate compared with the twofold decrease in nucleotide chain growth-rate observed after T4 infection ( Bremer & Yuan, 1968 a,b ) shows that there is a close correspondence in the rates of the two processes, as in exponentially growing cells; however, the rates of chain elongation and of mRNA decay decrease about twofold after infection.

Dependence of ribonucleic acid synthesis on continuous protein synthesis in yeast.

Using an auxotrophic strain of Saccharomyces cerevisiae, we examined the kinetics of ribonucleic acid (RNA) synthesis following inhibition of protein synthesis caused by amino acid starvation or cycloheximide. Removal of a required amino acid immediately stopped net protein synthesis. After a brief lag, RNA synthesis also ceased. Cycloheximide, a ribosome-inhibiting drug, also immediately halted net protein synthesis. Again RNA synthesis stopped after a brief lag. Although cycloheximide and amino acid starvation affect different steps in protein biosynthesis, both inhibited RNA synthesis in identical fashion. This indicates that amino acids do not play a unique role in the control of RNA production in rapidly growing yeast; rather, it suggests that RNA synthesis is responsive to the overall rate of protein synthesis itself.

Translational capacity of living frog eggs and oocytes, as judged by messenger RNA injection

Growing oocytes and activated eggs of Xenopus laevis have been injected with increasing amounts of 9 s haemoglobin messenger RNA extracted from rabbit reticulocytes. At low concentrations of injected RNA, there is a linear relationship between the amount of RNA injected and the amount of haemoglobin synthesized. Higher concentrations of RNA saturate the translational capacity of oocytes and eggs, so that increasing amounts of injected RNA fail to stimulate increased haemoglobin synthesis. Under these conditions, the rate of protein synthesis promoted by endogenous messenger RNA is unaffected by the injection of 9 s RNA and the over-all rate of protein synthesis by message-saturated cells is nearly doubled. At very high concentrations of injected RNA and after long periods of incubation, the injected RNA competes with endogenous messenger RNA for a translational component which limits the over-all rate of protein synthesis in injected cells. The results show that frog eggs and oocytes possess an unused capacity for translating injected messenger RNA, and that the amount of this spare capacity can be determined by the construction of messenger RNA saturation curves. The results also suggest that the normal rate of protein synthesis in these cells is limited by their content of functional messenger RNA.

The utilization of selenomethionine by Escherichia coli

The replacement of methionine by selenomethionine has been investigated in Escherichia coli. The analog is relatively non-toxic to growth of E. coli 26 at concentrations as high as 0.01 M, provided cysteine is added to the growth medium. dl-Selenomethionine, at 1·10 −4 M, causes a 95% reduction of methionine biosynthesis and is incorporated extensively into cellular proteins. The catalytic activity of β-galactosidase (EC 3.2.1.23) with 70–75% of its methionine residues replaced by selenomethionine, is the same as that of the unmodified enzyme. Although selenomethionine lengthens the generation time of E. coli 26, it has no effect on the overall rate of protein synthesis as measured by incorporation of [ 14C]phenylalanine, provided no amino acids (other than methionine) are limiting. Under the same conditions however, the rate of β-galactosidase induction drops 35% when selenomethionine is substituted for methionine. The analog causes significant inhibition of both bulk protein and β-galactosidase synthesis of amino acids other than methionine are also limiting. Selenomethionine also supports the growth of a methionine-requiring auxotroph derived from E. coli 26, but no growth occurred in ethionine, norleucine, or α-methylmethionine. The growth of E. coli K 12, however, is markedly inhibited by 1·10 −4 M dl-selenomethionine whether or not cyst(e)ine is present.

Effects of elevated temperatures on mengovirus ribonucleic acid synthesis and virus production in Novikoff rat hepatoma cells.

The production of mengovirus in Novikoff rat hepatoma cells is progressively reduced with an increase in incubation temperature of the cells from 34 to 40 C, in spite of the fact that about the same amounts of single-stranded and double-stranded viral ribonucleic acid (RNA) are synthesized at 34, 37, and 40 C; the rate of overall protein synthesis is as high at 40 C as at 37 C. At 40 C, progeny viral RNA accumulates in an undegraded form without being incorporated into virus particles. The results suggest that virus maturation is preferentially inhibited at supraoptimal temperatures. At 42 C, on the other hand, no viral RNA is produced and no viral RNA polymerase activity is detectable in cell lysates. Failure of infected cells to form viral RNA polymerase at 42 C is probably due to an impairment of protein synthesis since most of the polyribosomes are rapidly lost during incubation at 42 C and the rate of amino acid incorporation into protein is 70% lower at 42 C than at 37 C. When infected cells are shifted from 37 to 42 C during the period of active viral RNA synthesis, viral RNA polymerase activity is rapidly lost from the cells, and viral RNA synthesis ceases within 45 min. In contrast, the RNA polymerase is as active in vitro at 42 C as at 37 C, and the activity is relatively stable at 42 C.

Stringent Control of Transcription of Phage 80psu2

For about ten years now, we have known that the rate of synthesis of a specific protein (enzyme) in a bacterial cell is regulated mainly by controlling the rate of synthesis of the particular messenger RNA (mRNA) coding for the protein. We also know that bacterial cells have additional control mechanisms which regulate the overall rate of RNA synthesis relative to the overall rate of protein synthesis. In the extreme case, when protein synthesis stops (because a required amino acid is withdrawn), RNA synthesis stops immediately thereafter; this dependence of RNA synthesis upon continuing protein synthesis has been extensively studied and is known as “stringent control” (Stent and Brenner, 1961). Certain single-step mutations, which in E. coli occur at a single locus, abolish stringent control. These mutants are said to be “relaxed” for control of RNA synthesis since RNA synthesis continues despite the cessation of protein synthesis.

Structure and synthesis of a lipid-containing bacteriophage: II. Alterations in cytoplasmic and membrane-bound enzymes during replication of bacteriophage PM2

The specific activity of all membrane-bound enzymes studied decreased during infection of Pseudomonas BAL-31 with the lipid bacteriophage PM2. NADH oxidase, nitro blue tetrazolium chloride reductase, succinic dehydrogenase, and Mg 2+-dependent ATPase, all involved in oxidative phosphorylation, decreased in specific activity from the start of infection. Glucose phosphotransferase started to decrease rapidly in specific activity only at 10 min after infection (p.i.). Alkaline phosphatase, which presumably lies outside the cell membrane, decreased in a manner similar to the enzymes of oxidative phosphorylation, but not as much. In contrast, two cytoplasmic enzymes, hexokinase and glucose-6-phosphate dehydrogenase, increased in specific activity after infection, reaching a peak of twice the specific activity of uninfected cells at 30 min p.i. The overall rate of protein synthesis in control and infected cells was the same, at least until the commencement of cell lysis at about 60 min p.i. Whereas the bulk of cellular proteins labeled before infection was stable until about 60 min p.i., the specific radioactivity of prelabeled membrane-bound proteins decreased after infection. The latter observation led to the present working hypothesis: namely, that virus-specific proteins are inserted into the cellular membrane; this may or may not be accompanied by the displacement of some normal cellular membrane proteins. The altered cellular membranes, containing virus-specific proteins, may then form the outer shell of the virion.

Kinetics of viral deoxyribonucleic acid, protein, and infectious particle production and alterations in host macromolecular syntheses in equine abortion (herpes) virus-infected cells.

Infection of exponential-phase suspension cultures of mouse fibroblast cells (L-M) with equine abortion virus (EAV) resulted in inhibition of cell growth and marked alterations in host metabolic processes. The synthesis of deoxyribonucleic acid (DNA) and ribonucleic acid was inhibited within 4 hr after infection and was suppressed by more than 90% by the time of maximal virus replication (14 to 18 hr). The overall rate of protein synthesis, however, was similar in uninfected and virus-producing cells as determined by measurements of net protein and isotope incorporation. The time course of viral DNA and protein synthesis and assembly into mature virus was determined with the inhibitors 5-fluorodeoxyuridine (FUdR) and cycloheximide, respectively. Thus, viral DNA synthesis was essentially completed at 14 hr, and viral protein and infectious virus synthesis was completed at 18 hr. Although the number of plaque-forming units (PFU) produced by FUdR-treated cells (10(3) to 10(4) PFU/ml) was at least 3 logs less than that produced by untreated cells, the yield of physical particles (as determined by electron microscopy) was approximately the same at 30 hr after infection. Besides being relatively non-infective, the particles produced in FUdR-treated cells appeared morphologically incomplete as they contained little or no nucleoid material.

Wachstumsgeschwindigkeit der Peptidkette im Tabakmosaikvirus

The rate of incorporation of labelled amino acids into the complete tobacco mosaic virus (TMV), into soluble virus protein and into soluble cell proteins has been determined in discs of infected and healthy tobacco leaves. The rate of overall protein synthesis is increased by 50% in the infected leaves. At least 60% of the increase derives from the synthesis of virus-specific proteins and the synthesis of cellular proteins is not inhibited. The virus protein synthesis is strongly temperature dependent and shows a maximum at 28 °C. The exchange of free labelled amino acids between the external medium and the inner cellular pool reaches equilibrium within ten minutes. The influence of the exchange rate on the measurement of the kinetics of peptide chain synthesis is discussed in detail. Discs from infected leaves were incubated for short periods at low temperatures in media containing 3H-tyrosine or 3H-proline. Peptides isolated after 5 minutes incubation at 15 °C were found to be uniformly labelled with no apparent gradient of radioactivity from the N- to the C-terminus. The results indicate that the growth rate of the peptide chain at 15 °C is probably higher than 2 - 3 amino acids/sec and at 28 °C higher than 20 amino acids/sec. These values are higher than those for animal cells and similar to those for protein synthesis in Escherichia coli. Comparison of the growth rate of TMV protein with rate of total protein synthesis and the number of ribosomes in the tobacco leaves indicate that only a small portion of the ribosomes takes part in cell protein synthesis.

Embryonale Induktion und Hemmung der Ribonucleinsäure-Synthese durch Actinomycin D

Blastoporal lip (presumptive notochord and myotomes) together with adjacent ectoderm (which is induced to form the neural system by the blastoporal lip) from early gastrulae was explanted and treated with 0,5—2,5 µg/ml actinomycin D for periods ranging from two hours to several days. The RNA synthesis is completely inhibited at 2,5 μg/ml Actinomycin D and partially inhibited at 0,5 μg/ml actinomycin D. At 2,5 µg/ml some notochordal cells are still formed, but the differentiation of neural tissue is completely inhibited. At 0,5 μg/ml actinomycin D notochord and muscle are more resistent than the neural tissue. The inducing capacity of the blastoporal lip is not diminished after treatment with actinomycin D. Actinomycin D must therefore impair the competence of the ectoderm to be induced by inhibiting the RNA synthesis. If tissues from tailbuds, which are already differentiating into muscle, notochord and spinal cord are explanted and treated with actinomycin D the differentiation is partially inhibited, but a differential sensitivity of these tissues is not observed. The overall rate of protein synthesis is somewhat enhanced after treatment with 0,5 μg/ml actinomycin D for 2 — 5 hours.

Quantitative cytophotometric and autoradiographic studies on the rate of protein synthesis during interphase in mouse fibroblasts in vitro

An investigation was made of the rate of the overall protein synthesis during interphase growth in mouse fibroblasts in vitro (L-cells). Quantitative microspectrophotometric and microinterferometric methods were used for the determination of the cellular content of DNA, RNA and dry mass. The stage which the cell had reached in its interphase development was determined in two different ways. Firstly, the cell age, expressed in hr after the last cell division, was determined in a direct way by means of time-lapse cinematography. Secondly, the cell age was determined in an indirect way from the relationship between cell mass and relative cell age (the fraction of the total interphase time). This relation was derived from the mass distribution of individual cells in the growing fibroblast population. The amount of 14C-leucine, 3H-leucine and 35S-methionine incorporated into the cell protein after pulse incubation was quantitated by means of autoradiographic methods and used as a measure of the overall rate of protein synthesis in the cell. The absorption of tritium radiation in the cell was taken into account. The rate of incorporation of protein precursors into the growing cell increased over the whole of interphase by a factor of approximately 2. This result is in very strong agreement with previously published interferometric data on L-cells [17], which showed that the cellular dry mass also grew at an increasing rate over the whole of interphase by a factor of 2. The correlations between the number of grains over the cells and their DNA, RNA and mass content were investigated. The grain counts were found to have the best correlation with the cellular content of RNA and mass. These results support the idea that the total capacity for protein synthesis during interphase growth is controlled by the existing number of ribosomes in the cell.

Effect of mitomycin C on the synthesis of induced β-galactosidase in [formula omitted], [formula omitted

Addition of mitomycin C to E. , coli , B which had been induced for β-galactosidase by β-thiomethylgalactoside led to a temporary halt (2–3 minutes) of β-galactosidase synthesis followed by synthesis of this enzyme at slightly reduced rate. When the cells were pre-treated with mitomycin C and then induced by β-thiomethylgalactoside, the relative rate of induced β-galactosidase synthesis in treated versus normal cells was found to decrease with increasing duration of mitomycin C pretreatment even though the rate of overall protein synthesis was identical.