Studies on the control of ribosomal RNA synthesis in HeLa cells.

In many eucaryotic systems protein synthesis is coupled to ribosomal RNA synthesis such that shut-down of the former causes inhibition of the latter. We have investigated this stringency phenomenon in HeLa cells. The protein synthesis inhibitors cycloheximide and puromycin cause inactivation of both processes but valine starvation totally inhibits only the processing of 45-S RNA. DNA-dependent RNA polymerases from A, B and C (or I, II and III respectively) were extracted, separated partially by DEAE-cellulose chromatography and their activity levels determined. These do not decrease significantly during inhibition of protein synthesis. To find out whether or not form A is bound to its template under these conditions, proteins were removed from chromatin with the detergent sarkosyl. This does not affect bound RNA polymerase. Inhibition of protein synthesis caused up to 50% reduction in endogenous alpha-amanitin-insensitive chromatin-RNA-synthesising activity. This reduced level of activity was not affected by sarkosyl treatment. Levels in normal cells were stimulated. This result indicates that the form A RNA polymerase is not bound to its template when protein synthesis is inhibited.     

Dissociation of histone and DNA synthesis in x-irradiated HeLa cells

Although histone synthesis and DNA synthesis are normally very well coordinated in HeLa cells, their histone synthesis proved relatively resistant to inhibition by ionizing radiation. During the first 24 h after 1 000 R the rate of cellular DNA synthesis progressively fell to small fractions of control values while histone synthesis continued with much less relative reduction. Acrylamide gel electropherograms of the acid soluble nuclear histones synthesized by irradiated HeLa cells were qualitatively normal.     

RNA synthesis in permeable HeLa cells

The equilibria and kinetics are reported for the partial reactions of the catalytic cycle of the Ca²⁺ ionophore X537A in phospholipid vesicles. The analysis is based on the study of the behavior of the ionophore's intrinsic fluorescence in fluorescence lifetime, stopped-flow, temperature, and conventional steady-state fluorescence experiments. Binding to dimyristoyl phosphatidylcholine vesicles gives rise to an enhancement of the fluorescence. At the pH of study (7.4) this involves the singly negatively charged form (X^?). Complexation of the membrane-bound form (X_m^?) by monovalent (M⁺) or divalent (M²⁺) cations to give 1:1 (M-X)_m and (M-X)_m⁺ complexes, respectively, gives rise to a further fluorescence enhancement. No evidence could be found for stoichiometries other than 1:1 in the equilibrium experiments. The fluorescence of X537A in the presence of phosphatidic acid vesicles or phosphatidylcholine/ phosphatidylethanolamine or phosphatidylcholine/cholesterol mixtures is much smaller than for pure phosphatidylcholine. Fluorescence lifetime experiments show that this is due to a reduction in binding rather than a reduction of the quantum yield of the bound species. Fluorescence decay profiles from the above-mentioned membranes showed two exponential components indicating that there were two fluorescent species. The shorter-lived species had a lifetime of 3–5 ns and accounted for 80–90% of the membrane-bound ionophore. The longerlived species (9–13 ns) was estimated to account for the remaining 10–20%. This species enjoys a higher degree of hydrophobic shielding than the shorter-lived species. Possible interpretations in terms of the ionophore orientation in the membrane are discussed. Temperature-jump experiments show that the binding rate of the ionophore is fast. The binding and dissociation rate constants were ca. 2 × 10⁷m (PC)^?1 s^?1 and 2 × 10³ s^?1, respectively. Stopped-flow experiments gave evidence for a slower “insertion” process with a ca. 10-ms half-time. Analysis shows that this process is capable of transport of (K-X) across the membrane with a rate constant ≤ 69^?1. In the presence of divalent cations a slower process involving transport of M²⁺-ionophore complexes across the membrane can be observed. The dependence of the rate on the total ionophore concentration indicates that the transported species is a neutral (M-X₂) complex. The lower limit for the rate constant for transport of the (Ca-X₂) complex is 35 s^?1. The divalent cation specificity of the overall reaction was shown to be Mg²⁺ ? Ca² < Sr²⁺ < Ba²⁺. The rates of the overall transport at low ionophore concentration are limited by the equilibrium constant for formation of the (M-X₂)_m complex from the (M-X)_m⁺ complex.     

Adenylate-rich oligonucleotides of ribosomal and ribosomal precursor RNA from HeLa cells

HeLa cell ribosomal precursor (45S) RNA has been found to contain nucleotide sequences also found in 18S RNA and others found in 28S RNA. Thus, 18S RNA and 28S RNA are readily distinguishable, whereas 45S RNA is similar to 18S and 28S RNA combined. The nucleotide sequences analysed were those resistant to the combined action of pancreatic and T1 ribonucleases.     

Coordination of ribosomal RNA synthesis in vertebrate cells

Xenopus embryo cells and HeLa cells were investigated under various conditions to test for coordinate synthesis of high molecular weight (28S and 18S) and low molecular weight (5S) rRNA. Xenopus embryos initiate 28S and 18S rRNA synthesis at gastrulation (Brown and Littna, '64); we found that 5S rRNA synthesis is coordinately initiated with the 28S and 18S rRNAs at the same time in development. Dissociated Xenopus blastula cells were cultured in vitro for several hours to condition the medium; post-gastrula cells were then grown in the conditioned medium to test for the existence of an inhibitor of rRNA synthesis. No inhibitor was detected. Low doses of actinomycin D profoundly inhibit the synthesis of 28S and 18S rRNA in HeLa cells, while 5S rRNA synthesis is less affected by this treatment. Therefore, actinomycin D does not produce a coordinate inhibition of all rRNA species. Similar effects of the antibiotic were found in cultured amphibian cells. Synchronized HeLa cells reinitiating RNA synthesis following mitosis also respond to actinomycin D in a non-coordinate manner.     

Reversible inhibition of ribosomal RNA synthesis in HeLa by lucanthone (Miracil D) with continued synthesis of DNA-like RNA

Ribosomal RNA synthesis was selectively inhibited in HeLa cells by lucanthone, a clinically useful schistosomicide which shares many of the properties of Actinomycin D. Synthesis of DNA-like RNA continued during complete inhibition of ribosomal RNA synthesis. Under these conditions newly synthesized DNA-like RNA accumulated normally in polyribosomes of the cell cytoplasm; most of it appeared to be messenger RNA. DNA synthesis was partially inhibited by lucanthone but protein synthesis was undisturbed. Synthesis of ribosomal RNA promptly resumed after removal of lucanthone and cell survival was not affected if exposures to the drug were limited to two hours.     

Inhibition of exogenous RNA-dependent protein synthesis by a low-molecular-weight RNA from nuclear ribonucleoprotein particles of adenovirus-infected HeLa cells

Low-molecular-weight RNA (4S to > 5.5S) isolated from nuclear ribonucleo-protein particles of adenovirus-infected HeLa cells inhibited cell-free protein synthesis directed by polyribosomal RNA from rabbit reticulocytes by more than 80%. In a reconstituted system inhibitory RNA did not prevent the binding of Met-tRNA_f-GTP-IF ternary complex to 40S subunits; however, it repressed the formation of 80S from 40S-mRNA complex and 60S subunits. In binding assays in which authentic IF-M2A and IF-M2B were present, the inhibitor competed with messenger molecules for binding site(s) in IF-M2B. The inhibitory RNA appears to be a 5.5S RNA.