Physiology of rat-liver polysomes: Protein synthesis by stable polysomes

Certain qualitative aspects of protein synthesis in the livers of starved, starved-re-fed and actinomycin D-treated rats have been examined by polyacrylamide-gel electrophoresis. Animals were exposed to a mixture of (14)C-labelled acids for 18-20min. and killed, and an ultrasonic extract of newly formed protein in microsomal vesicles was prepared and examined by gel electrophoresis. In normal and starved-re-fed animals, 27% of the newly synthesized protein was albumin. During starvation, when RNA synthesis was decreased, the percentage of newly formed protein as albumin rose. After actinomycin D treatment of starved-re-fed rats, when only stable messenger RNA persisted in the cytoplasm, albumin synthesis increased to 63% of the total. This finding suggested that albumin was the primary protein synthesized on stable messenger RNA.     

The effect of feeding with a tryptophan-free amino acid mixture on rat-liver polysomes and ribosomal ribonucleic acid

1. Starving rats were given complete and tryptophan-deficient amino acid mixtures by stomach tube and were killed from 1 to 7hr. later. The polysome profile in the livers of rats fed with the tryptophan-deficient mixture showed a shift in distribution such that the large aggregates were decreased and the small aggregates were increased, particularly dimers. This polysome shift was reversed when the complete amino acid mixture was given by stomach tube 2hr. after administering the tryptophan-free amino acid mixture. 2. After removal of liver polysomes by centrifugation, some smaller ribosomal aggregates (oligosomes) remaining in suspension were harvested by prolonged centrifugation of the supernatant fluid. A large increase in the dimer population of this fraction was observed in the rats receiving the incomplete mixture. 3. When the polysome and oligosome fractions were incubated with cell sap, an energy-generating system and labelled amino acids dl-[1-(14)C]leucine and l-[Me-(14)C]tryptophan were incorporated into the cell fractions in the ratio 4.5:1. Preparations of polysomes and oligosomes from rats fed with the tryptophan-free amino acid mixture showed a decreased amino acid-incorporating activity compared with particulate preparations made from rats fed with the complete mixture. 4. The yield of free ribosomes prepared from the unfractionated liver microsomes by treatment with iso-octane was 40-50% greater in rats fed with the amino acid mixture deficient in tryptophan. 5. A post-microsomal fraction was prepared from cell sap and was shown to consist of ribosomal sub-units. When the animals were fed with the tryptophan-deficient mixture, there was an increase in content of this post-microsomal fraction and in the ratio 30s RNA/19s RNA. Rats were also given [5-(3)H]orotic acid at the time of feeding with the amino acids. Lack of tryptophan in the mixture caused a decrease in the specific activity of both RNA fractions which affected the 30s RNA more extensively than the 19s RNA. 6. These changes in the distribution and quantity of the cellular components engaged in protein synthesis are discussed in relation to RNA metabolism and amino acid-incorporating activity of the liver cell and their response to feeding with the tryptophan-free amino acid mixture.     

Isolation and characterization of poly(adenylic acid)-containing messenger ribonucleic acid from rat liver polysomes

Undegraded rat liver polysomes were obtained after homogenizing the tissue in a medium containing NH4Cl, heparine, and yeast tRNA. Purification of poly(A)-containing RNA from polysomal RNA was accomplished by affinity chromatography on oligo(dT)-cellulose columns. Poly(A)-containing RNA molecules were monitored by the formation of ribonuclease-resistant hybrids with [3H]poly(U). To improve the separation of messenger RNA and ribosomal RNA by oligo(dT)-cellulose it was found essential to dissociate the aggregates formed between both molecular species by heat treatment in the presence of dimethylsulfoxide (Me2SO) prior to chromatography. Sucrose gradient analysis under denaturing conditions showed that the preparations obtained were virtually free of ribosomal RNA. Poly(A)-containing RNA constituted approx. 2.2% of the total polysomal RNA and the number average size was 1500--1800 nucleotides, as judged by sedimentation analysis on sucrose density gradients containing Me2SO. Approximately 8.2% of the purified preparation obtained was able to anneal with [3H]poly(U); the number average nucleotide length of the poly(A) segment of the RNA population was calculated to be 133 adenylate residues. Based on these values, our preparations appear to be greater than 90% pure. The RNA fractions obtained after oligo(dT)-cellulose chromatography were used to direct the synthesis of liver polypeptides in a heterologous cell-free system derived from wheat-germ. The system was optimized with respect to monovalent and divalent cations, and presence of polyamines (spermine). More than 65% of the translational activity present in the unfractionated polysomal RNA was recovered in the final poly(A)-containing RNA fraction. However, about 25% of the activity was found to be associated with the unbound fraction which was essentially free of poly(A)-containing RNA. Immunoprecipitation analysis with a specific antiserum to rat serum albumin demonstrated that about 6--8% of the labeled synthetic products translated from the poly(A)-containing RNA sample corresponded to serum albumin. Analysis of the translation products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a heterogeneous distribution of molecular sizes ranging from 15 000 to greater than 70 000 daltons. Spermine not only increased the overall yield and extent of protein synthesis, but also resulted in higher yields of large protein products. Under optimal translation conditions a discrete peak representing about 7% of the total radioactivity was observed to migrate with rat serum albumin.     

In vivo stability of bacteriophage T4 messenger ribonucleic acid

Cohen, Paul S. (St. Jude Children's Research Hospital, Memphis, Tenn.), and Herbert L. Ennis. In vivo stability of bacteriophage T4 messenger ribonucleic acid. J. Bacteriol. 92:1345-1350. 1966.-A mutant of Escherichia coli B, defective in its transport and concentration of K(+), synthesizes ribonucleic acid (RNA) without the simultaneous synthesis of protein when depleted of this cation. The mutant was used to study the in vivo stability of phage T4 messenger RNA (mRNA) in the presence and absence of K(+). Experiments were performed in which the turnover of phage T4 mRNA was determined in infected cells continuously synthesizing RNA and in cells in which RNA synthesis was inhibited by actinomycin D. Phage mRNA was found to be more stable in the absence of K(+) than in the presence of either the cation or chloramphenicol.     

Distribution of messenger ribonucleic acid in polysomes and nonpolysomal particles of sea urchin embryos: translational control of actin synthesis

We have used cell-free translation and two-dimensional gel electrophoresis to examine the complexities of the polysomal and cytoplasmic nonpolysomal [ribonucleo-protein (free RNP)] messenger ribonucleic acid (mRNA) populations of sea urchin eggs and embryos. We show that all species of mRNA detected by this method are represented in both the polysomes and free RNPs; essentially all messages present in polysomes are also in the free RNP fraction. However, the cytoplasmic distribution is clearly nonrandom since some templates are relatively concentrated in the free RNPs and others are predominantly in the polysomes. The polypeptides synthesized under the direction of unfertilized egg mRNA are qualitatively indistinguishable from those made by using embryonic mRNA, indicating that the complexity of the abundant class mRNA remains unchanged from egg through early development. However large changes in the abundancies of specific mRNAs occur, and changes are detected in the polysomal/free RNP distribution of some mRNAs through development. The differences in the realtive abundancies of specific mRNAs between polysomes and free RNPs and the developmental changes that take place indicate significant cytoplasmic selection of mRNA for translation. Three different forms of actin (termed alpha, beta, and gamma) were identified among the translation products. Messages for all three are present in the unfertilized egg and early cleavage embryo, yet the gamma form is preferentially located in the polysomes and the alpha and beta in the free RNPs. The relative concentrations of the three change greatly during development as do their relative distributions into polysomes and free RNPs. Examinations of in vivo labeled proteins largely support the in vitro findings. The results indicate that the synthesis of actin mRNAs increases greatly during development and that the expression of the actin mRNAs is partly controlled at the translation level during early development.     

Lifetime of bacterial messenger ribonucleic acid

Moses, V. (University of California, Berkeley), and M. Calvin. Lifetime of bacterial messenger ribonucleic acid. J. Bacteriol. 90:1205-1217. 1965.-When cells from a stationary culture of Escherichia coli were placed in fresh medium containing inducer for beta-galactosidase, growth, as represented by increase in turbidity and by total protein synthesis, started within 30 sec. By contrast, beta-galactosidase synthesis was greatly delayed compared with induction during exponential growth. Two other inducible enzymes (d-serine deaminase and l-tryptophanase) and one repressible enzyme (alkaline phosphatase) showed similar lags. The lags were not due to catabolite repression. They could not be reduced by pretreatment of the culture with inducer, or by supplementing the fresh medium with amino acids or nucleotides. The lag was also demonstrated by an i(-) mutant constitutive for beta-galactosidase synthesis. An inhibitor of ribonucleic acid (RNA) synthesis, 6-azauracil, preferentially inhibited beta-galactosidase synthesis compared with growth in both inducible and constitutive strains. Puromycin, an inhibitor of protein synthesis, acted as an inhibitor at additional sites during the induction of beta-galactosidase synthesis. No inhibition of the reactions proceeding during the first 20 sec of induction was observed, but puromycin seemed to prevent the accumulation of messenger RNA during the period between 20 sec and the first appearance of enzyme activity after 3 min. It is suggested that these observations, together with many reports in the literature that inducible enzyme synthesis is more sensitive than total growth to some inhibitors and adverse growth conditions, can be explained by supposing that messenger RNA for normally inducible enzymes is biologically more labile than that for some normally constitutive proteins. The possible implications of this hypothesis for the achievement of cell differentiation by genetic regulation of enzyme synthesis are briefly discussed.