The effect of puromycin on intranuclear steps in ribosome biosynthesis.

Inhibition of protein synthesis by puromycin (100 gamma/ml) is known to inhibit the synthesis of ribosomes. However, ribosomal precursor RNA (45S) continues to be synthesized, methylated, and processed. Cell fractionation studies revealed that, although the initial processing (45S --> 32S + 16S) occurs in the presence of puromycin, the 16S moiety is immediately degraded. No species of ribosomal RNA can be found to have emerged from the nucleolus. The RNA formed in the presence of puromycin is normal as judged by its ability to enter new ribosomal particles after puromycin is removed. This sequence of events is not a result of inhibition of protein synthesis, for cycloheximide, another inhibitor of protein synthesis, either alone or in combination with puromycin allows the completion of new ribosomes.

Comparison of the relative synthesis of the proteins of the 50S ribosomal subunit in growing and valine-deprived HeLa cells.

Quantitative studies of the synthesis of the ribosomal proteins of the 50S ribosomal subunit have been made with growing versus valine-deprived HeLa cells. The synthesis of total cell protein and 50S subunits was also compared between the growing and nongrowing cells. It was found that between 12 and 20 hr of valine deprivation the net synthesis of 50S subunits drops to approximately 8% of that in control cells while the over-all synthesis of 50S subunit ribosomal proteins declines to approximately 12% of that in the controls. However, the synthesis rates for each of two particular 50S subunit proteins decline to approximately 8% of the rates in the growing cells, indicating that the synthesis of one of these proteins may be rate limiting for 50S subunit biosynthesis in valine-deprived HeLa cells. Other evidence indicates that the regulation of synthesis of the ribosomal proteins in valine deprivation depends on control at the level of transcription or translation rather than being a function of the relative valine content of these proteins.

The turnover of nuclear DNA-like RNA in HeLa cells.

The subcellular distribution of various types of RNA in HeLa cells is described. In addition, the relative rate of synthesis of the major classes of nuclear RNA has been determined. From these experiments it can be deduced that the heterogeneous nuclear RNA fraction is rapidly synthesized and degraded within the cell nucleus.

Influence of valine deprivation and its reversal on fatty acid metabolism in HeLa cells.

The effects that amino acid starvation and re-supplementation have on fatty acid metabolism in HeLa cells have been studied using radio gas chromatographic techniques. Deprivation of valine for 13.5 h caused fatty acid de novo biosynthesis, elongation and desaturation to cease. This effect was reversed within 5 h by adding valine back to the culture. During deprivation accumulation of triacylglycerol occurred. The return of valine to the culture caused compositional changes in the triacylglycerols and phosphatidylcholines.

40S subunit X Met-tRNAf complexes and initiation factor eIF-2 phosphorylation in mammalian cells accumulating uncharged tRNA.

A significant, reversible decline in the level of 40S subunit X Met-tRNAf complexes was observed in HeLa cells treated with either of two inhibitors of tRNA charging, histidinol or O-methylthreonine. A decline in 40S X subunit Met-tRNAf complexes was also seen in HeLa cells deprived of histidine and in mutant Chinese hamster ovary cells, bearing a temperature-sensitive leucyl-tRNA charging enzyme, when they were incubated at a non-permissive temperature. Treatment with histidinol or O-methylthreonine did not affect the relative degree of phosphorylation of the alpha subunit of initiation factor eIF-2. Histidinol did not stimulate the activity of the endogenous kinase for eIF-2 alpha subunit in extracts prepared from HeLa cells. In the presence of histidinol the phosphorylation level of a 25000-dalton protein was reversibly increased.

Synthesis of proteins and RNA of the 60S ribosomal subunit in HeLa cells recovering from valine deprivation.

The synthesis of ribosomes in HeLa cells was studied during recovery from a 20-hour deprivation for valine. The rates of incorporation of labeled precursors into ribosomal pre-RNA, processed rRNA, total cellular proteins, and proteins of the 60S ribosomal subunit returned to normal or nearly normal levels immediately after restoration of valine to the medium. Specific proteins of the 60S ribosomal subunit, whose apparent net synthesis is reduced more than that of the other proteins of the 60S ribosomal subunit during valine deprivation, were no longer undersynthesized after valine was restored. This rapid recovery suggests that the apparent decrease in the net rate of synthesis of these ribosomal proteins during valine deprivation is effected at the translational or post-translational level. No evidence of significant synchrony in any particular stage of the cell cycle was observed after a 20-hr valine deprivation.

Isolation and characterization of a cycloheximide-resistant mutant of Acanthamoeba castellanii Neff.

A cycloheximide-resistant mutant was isolated from the amoeba Acanthamoeba castellanii Neff. Drug resistance was found to be due to a ribosomal modification.

Regulation of protein synthesis initiation in HeLa cells deprived of single essential amino acids.

In HeLa cells deprived of valine, histidine, or methionine initiation of protein synthesis decreases rapidly and disaggregation of polyribosomes also occurs. The mechanism of inhibition does not seem to involve the supply of RNA in the cell, and thus it differs from the initiation of inhibition at elevated temperatures. Polyribosomes rapidly form again if the missing amino acid is restored, even in the presence of actinomycin D.

Isolation, characterization, and stability of the 30S ribosomal RNA complex from HeLa cells.

The HeLa 30S rRNA molecule (historically designated 28S rRNA) can be dissociated into two components, a 7S rRNA and a large rRNA component which we call 29S rRNA. To evaluate conformational differences between the 30S rRNA complex and the isolated 29S rRNA component of the complex, viscosity, sedimentation velocity, circular dichroism, and ultraviolet absorption measurements with the two species were performed. Sedimentation equilibrium studies were also carried out with the 30S rRNA complex. In addition, the kinetics of the reaction which dissociates the 30S rRNA complex were characterized. The removal of glycogen-like molecules by cetyltrimethylammonium bromide prescipitation of the rRNA and the preequilibration of rRNA with solvent by Sephadex column chromatography were found to be essential for reproducibility. The s20,2o values for the 30S rRNA complex and the isolated 29S rRNA were determined from the experimental data obtained at various rRNA concentrations as 29.89 plus or minus 0.40 and 29.09 plus or minus 0.14, respectively. The corresponding intrinsic viscosity values were 74 plus or minus 5 and 67 plus or minus 5 cm3/g, respectively. The optical properties of the 30S rRNA and 29S rRNA were not significantly different. These results indicate that there is no significant conformational difference between 30S rRNA and 29S rRNA under the conditions studied. We conclude from the sedimentation equilibrium data that the molecular weight of 30S rRNA is 2.1 x 10-6. From the kinetic data, the 30S rRNA dissociation appears to be an irreversible, cooperative, and ionic strength dependent reaction which at an ionic strength of 0.051 has an activation enthalpy of 123.5 kcal/mol and an activation entropy of 0.21 kcal/(mol deg).

Polyadenylic acid sequences in the heterogeneous nuclear RNA and rapidly-labeled polyribosomal RNA of HeLa cells: possible evidence for a precursor relationship.

Polyadenylate sequences have been found covalently linked in heterogeneous DNA-like nuclear RNA of HeLa cells. This poly(A) material seems homogeneous in size and accounts for about 0.5% of such RNA. Similar poly(A) sequences were found in rapidly-labeled polyribosomal RNA, thought to be messenger RNA. A possible model for mRNA synthesis from large heterogeneous nuclear RNA precursor molecules is discussed.

Mechanism of action of the mycotoxin trichodermin, a 12,13-epoxytrichothecene.

Trichodermin is a member of a group of closely related compounds-the 12,13-epoxytrichothecenes-that form a medically and economically important class of mycotoxins produced by fungi that spoil fruit and grain. Our studies show that trichodermin is a very potent inhibitor of protein synthesis in mammalian cells. Since ribosomes remain in polyribosomes in inhibited cells, trichodermin inhibits the elongation and/or termination processes of protein synthesis. In vitro, trichodermin is a potent inhibitor of the peptidyl transferase activity required for elongation and/or termination. An in vitro comparison of the effects of three peptidyl transferase inhibitors on elongation and termination indicates that anisomycin acts primarily on elongation while trichodermin and sparsomycin act primarily on termination. A new in vivo test to distinguish elongation inhibitors from termination inhibitors confirms that trichodermin inhibits primarily the termination process. Thus trichodermin inhibits protein synthesis by blocking the activity of peptidyl transferase required for termination. These studies suggest that the toxicosis caused by one of the 12,13-epoxytrichothecenes is due to its action as a protein synthesis inhibitor involving the peptidyl transferase activity of the eukaryotic ribosomes.