Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis最新文献

The incorporation of ³²P_i into the ribosomal protein S6, which is a component of the 40 S ribosomal subunit, was measured in Ehrlich ascites tumor cells growing in suspension culture. During a 2-h incubation period, incorporation was 10-fold greater into S6 of cells growing exponentially in complete medium than in cells in which protein synthesis and growth were inhibited by omission of glutamine from the medium. Since the labeling of other phosphoproteins was not similarly depressed in glutamine-deprived cells, the decreased labeling most likely reflects a specific decrease in phosphate incorporation into S6 rather than a lower phosphate pool specific activity. The cycling of ribosomes is inhibited in glutamine-deprived cells, with an accumulation of monomeric ribosomes indicating an inhibition of protein synthesis initiation. To assess whether the decreased phosphorylation of S6 in the ribosomes of glutamine-deprived cells was responsible for their decreased ability to initiate protein synthesis and enter polyribosomes, a mixture of ¹⁴C-labeled ribosomes from rapidly growing cells and ³H-labeled ribosomes from glutamine-deprived cells was added to a rabbit reticulocyte lysate cell-free protein-synthesizing system in which ribosomes cycle actively. The ${}^{14}\text{C}{}^3\text{H}$ ratio was determined in polyribosomes as a measure of the relative ability of the two kinds of ribosomes to initiate protein synthesis. The ${}^{14}\text{C}{}^3\text{H}$ ratio in polyribosomes was found to be identical to the input of labeled ribosomes, indicating an equal ability of ribosomes from growing cells and glutamine-deprived cells to function in initiation. Control studies indicated lack of significant dephosphorylation of S6 during purification of ribosomes and during incubation in the reticulocyte lysate. Thus, a functional difference between more and less phosphorylated ribosomes could not be demonstrated in this system.

The effect of some ionophores and metabolic inhibitors on reticulocytes protein synthesis was examined. At μM concentrations, valinomycin, nigericin and CCCP rapidly inhibit protein synthesis, while with antimycin-A or DCCD the inhibition is rather slow. The onset of the arrest of protein synthesis coincides with a 20–30% drop in the intracellular ATP content. No inhibition in protein synthesis or drop in ATP was found after 1 h incubation without glucose or in the presence of 2-deoxyglucose. It is shown that the inhibition by valinomycin, nigericin or CCCP is not due to their effect on K⁺ and/or H⁺ fluxes through the plasma membrane. Reticulocytes incubated at pH 8.2 show much lower inhibition of protein synthesis by nigericin, CCCP, DCCD or antimycin-A. On the other hand, at this alkaline pH, starvation to glucose causes high inhibition of protein synthesis. It is concluded that the ionophores inhibit protein synthesis due to their uncoupling effect on mitochondrial ATP synthesis. At high pH, the glycolytic activity is relatively high, and the ATP generated by the glycolysis can compensate to some degree for the ATP loss in the oxidative phosphorylation.

Epithelial and mesenchymal dental cells were grown in primary monolayer culture and the ability of both cell types to synthesize interstitial collagens was investigated. Pepsin-solubilized collagens were analyzed by CM-cellulose chromatography and both cell types were found to synthesize collagen type I, type III and type I trimer. The collagen phenotype of mesenchymal cells (type I: 82.4%, type III: 8.5%, type I trimer: 9.1%) was different from that of epithelial cells (type I: 71.8%, type III: 9.5%, type I trimer: 18.7%). The radioactivity incorporated into collagen molecules by mesenchymal cells was 34-times greater than the radioactivity incorporated by epithelial cells. This result agreed with previous observations obtained from tissue culture experiments (Lesot, H. and Ruch, J.V. (1979) Biol. Cell. 34, 23–37) which indicated a low synthesis of interstitial collagens by isolated dental epithelia when compared to isolated dental mesenchymes.

Use of Escherichia coli RNA polymerase for in vitro transcription of chromatin results in the formation of double-stranded RNA molecules, which consist of a strand of endogenous mRNA and a complementary strand of de novo synthesized RNA. Unless the duplex structures are dissociated prior to isolation of the in vitro transcripts on sulfhydryl agarose columns, the endogenous mRNA can result in over-estimates of in vitro gene-specific transcription. Substitution of wheat germ RNA polymerase B for the bacterial enzyme overcomes this artifact. When mouse fetal liver chromatin is used as template, most of the mRNA synthesized by the plant enzyme is in a single-stranded form. More importantly, this synthesis is directed by a DNA template. Hybridization studies suggest that in vitro transcription of chromatin with wheat germ RNA polymerase B maintains some fidelity to genetic restrictions which operate in vivo.

Arginine-rich histones H2A, H2B, H3 and H4 contain two regions of interaction with cyclic nucleotide-dependent protein kinases: a substrate phosphorylation site and a region which noncompetitively inhibits cyclic nucleotide binding to the protein kinases. We have compared the interaction of cyclic nucleotide-dependent protein kinases with these two sites in histones which are organized in nucleosome structures with the interaction of the enzymes with free histones. Whereas histones in solution are readily phosphorylated by cyclic GMP-dependent protein kinase and the catalytic subunit of cyclic AMP-dependent protein kinase, mononucleosomes are not phosphorylated by these enzymes. Histones extracted from mononucleosomes can be phosphorylated, indicating that the lack of phosphorylation of nucleosomes is not due to covalent modification of the histones but to their organization within the nucleosome structure. Whereas histones in solution are effective noncompetitive inhibitors of cyclic GMP binding to cyclic GMP-dependent protein kinase and of cyclic AMP binding to the regulatory subunits of cyclic AMP-dependent protein kinase, mononucleosomes do not affect cyclic nucleotide binding. These studies indicate that histones which are organized in nucleosome structures are neither substrates nor modifiers of cyclic nucleotide-dependent protein kinases.

Single-stranded DNA is not transcribed randomly by yeast RNA polymerase B. A denatured yeast DNA fragment, containing the gene for yeast alcohol dehydrogenase I, directs the transcription of defined RNA products visualized as discrete RNA · DNA hybrid bands following S₁ nuclease treatment and agarose gel electrophoresis. Blocking the 3′ end of the template by 3′ deoxyadenosine did not change the band pattern but reduced the proportion of RNA covalently bound to the DNA from 20 to 4%. On the other hand, the band pattern was affected by the salt concentration, the nature of the divalent cation and the nucleoside triphosphate concentration. The four major RNA bands, found at low substrate concentration, hybridized to the same region of the template. This observation suggests the potential requirement for DNA destabilization in gene activation.

The effect of ATP on Escherichia coli DNA polymerase I has been investigated as a function of the concentration of substrates and divalent metal ions in the presence of activated DNA as template. At saturating Mg²⁺ concentration 1.5 mM ATP stimulated 2.5-times the incorporation of [³H]dGTP when only one substrate (dGTP) was present, and had no significant effect in the presence of all four dNTPs, whereas under similar conditions, a saturating concentration of dATP increased the reaction rate only 1.5-times. At optimal Mn²⁺ concentrations ATP also showed a similarly marked effect only in the case when one substrate (dGTP) was present in the reaction. The optimal concentration of Mn²⁺ was shifted by ATP to higher concentrations both in the presence of one and of all four substrates. ATP did not influence the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for dGTP, while $\text{V}$ was increased by a factor of about 2.5. The possible presence of dNTP in ATP, as inpurity, was ruled out by isotope dilution analysis. Thus, ATP stimulated the polymerization reaction only under limited conditions, i.e., when one substrate was present in the reaction.

An analogue of valine in which valine is coupled by its amino group to a chlorinated dihydroisocoumarin moiety inhibits competitively the tRNA^Val charging with valine catalyzed by pure yeast valyl-tRNA synthetase. The inhibition constant is much lower for the ATPaiPP_i exchange reaction than for the tRNA charging one, indicating different rate-determining steps in the two reactions.

A method for large-scale isolation of streptomycete ribosomal subunits involving centrifugation in hyperbolic sucrose density gradients in a zonal rotor was developed. Ribosomal proteins were extracted from 30 S and 50 S subunits of Escherichia coli A19 and primary mycelium of Streptomyces granaticolor. Their two-dimensional electropherograms differed considerably. Purified 30 S and 50 S subunits from S. granaticolor mycelium contained 21 and 36 ribosomal proteins, respectively. Only 8 proteins in the mycelial ribosomes possessed identical electrophoretic mobilities as corresponding E. coli ribosomal proteins, viz., S4, S12, S16, L1, L2, L14, L16 and L19. Despite the differences in physico-chemical properties, functional correspondence is likely to exist between certain ribosomal proteins from the two bacteria. The range of molecular weights of vegetative S. granaticolor ribosomal proteins was similar to that in other prokaryotes. Ribosomal proteins were further isolated from 70 S ribosomes of S. granaticolor dormant spores. The spore ribosomal proteins differed markedly from those of the primary mycelium and their total number was lower. The ribosomal protein alterations are presumed to take part in the regulation of the streptomycete cell differentiation.

Estrogens are involved in the stimulation of prolactin synthesis in the rat anterior pituitary. After 5 days of treatment with 17β-estradiol, strong enhancement of [³H]leucine incorporation into prolactin and stimulation of translatable preprolactin mRNA, respectively, were noted. Increase in prolactin synthesis was also found following daily application with estriol and the synthetic estrogen 1,3-dibenzoyloxy-17β-methyl-1,3,5(10)-estratrien-17β-ol (DB-EE2). The antiestrogen tamoxifen (trans-1-(p-β-dimethylaminoethoxyphenyl)-1,2-diphenylbut-1-ene) was demonstrated to inhibit estradiol-stimulated prolactin synthesis. Antagonistic effects of tamoxifen were dose dependent. Low doses of the antiestrogen were already sufficient to suppress 17β-estradiol-enhanced levels of prolactin mRNA. On the other hand, estriol potentiated 17β-estradiol-stimulated levels of serum prolactin and prolactin synthesis. Our results add further information about the transcriptional control by estrogens and demonstrate inhibitory actions of antiestrogens on distinct regulatory levels of protein synthesis in the rat pituitary.