The effect of some ionophores and metabolic inhibitors on reticulocytes protein synthesis was examined. At microM 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.
A pyrimidine nucleoside phosphorylase was partially purified from human blood platelets. The purified enzyme, as well as crude enzyme preparations, catalyses the phosphorolysis of thymidine and deoxyuridine, but not of uridine, and is able to catalyse direct pentosyl transfer from these deoxyribonucleosides to uracil or thymine; this enzyme has the properties of a thymidine phosphorylase. It has a molecular weight of about 110 000 and is composed of two identical subunits; it is phosphate dependent, has a maximal activity at a pH value of 5.7, and an isoelectric point of 4.4. This enzyme was mainly of cytoplasmic origin. Although platelet thymidine phosphorylase could promote the degradation or synthesis of thymidine, intact platelets degraded thymidine but were not able to synthesize thymidine from thymine. Blood platelets may play an important role in the degradation of plasma thymidine.
In a poly(U)-programmed translation system, neomycin stimulates the misincorporation of tyrosine and of serine which, according to Thompson and Stone (Thompson, R.C. and Stone, P.J. (1977) Proc. Natl. Acad. Sci. USA. 74, 198-202), are normally rejected at an initial discrimination step during the binding of charged tRNAs to the ribosome. In contrast, streptomycin favors the misincorporation of isoleucine which is normally rejected at a subsequent GTP-dependent discrimination step, the so-called proofreading step. The labeling of the ribosome with N-ethylmaleimide mimics the effect of streptomycin in that it stimulates the misincorporation of isoleucine but not of tyrosine or serine. This effect is correlated with the labeling of protein S18 but not with that of protein S1. These observations indicate that the sulfhydryl group of protein S18 is located within a ribosomal domain involved in the proofreading control of tRNA selection. Taking into account our previous results that streptomycin and neomycin perturb ribosomal areas around the sulfhydryl groups of proteins S18 and S1, respectively, we suggest that these antibiotics induce misreading by different mechanisms which are linked to such perturbations.
The methylation patterns produced in Escherichia coli B tRNA by a range of concentrations of the weak carcinogen dimethyl sulphate were examined with the following results: 1. 1,7-Dimethylguanosine was found to be formed in high amounts in the tRNA methylation reaction at high concentrations of methylating agent. 2. The dialkylated compound was recovered mainly in the form of derivatives, the spectral and chromatographic behaviour of which varied according to the procedures used for their isolation. Similar results were obtained for the in vivo methylation of rat-liver tRNA: after administration of a very high dose of the powerful carcinogen dimethylnitrosamine, 1,7-dimethylguanosine was found in rat-liver tRNA. Moreover, the analysis of the time-course of nucleic acid methylation indicated that this dialkylated product was still present in rat-liver tRNA when the major product of alkylation, 7-methylguanine, had almost completely disappeared.
A formylmethionyl-tRNAf deacylase has been purified about 330-fold from a crude initiation factor preparation (1 M NH4Cl ribosomal wash) from Escherichia coli Q13. The enzyme was nearly homogeneous and had an apparent molecular weight of 24 000. Rat liver methionyl-tRNAf and E. coli methionyl-tRNAm were not hydrolyzed significantly by the enzyme under standard conditions. Q beta RNA- and AUG(A)n-directed polypeptide synthesis was inhibited by the enzyme. The inhibition was at the level of initiation of polypeptide synthesis. The enzymatic activity was inhibited by various factors necessary for polypeptide synthesis. The activity was inhibited more by NH4Cl and spermidine than by Mg2+, GTP and ATP. The complex of formylmethionyl-tRNAf, initiation factor 2 and GTP was resistant to enzymatic hydrolysis, and the resistance was enhanced by the addition of AUG and ribosomes to the above reaction mixture.
We have shown that pyridoxal 5′-phosphate is an effective inhibitor of Rauscher leukemia virus DNA polymerase (Biochemistry 15 (1976) 3620). Detailed studies of this inhibition revealed that, in addition to the phosphate and aldehyde groups of pyridoxal phosphate, the presence of a divalent cation is essential for the inhibitory action. The synthesis directed by template primers containing GC base-pairs exhibited more resistance to pyridoxal phosphate inhibition than did that directed by AT base-paired templates. Maximal inhibitory activity of pyridoxal phosphate, however, is noted in the presence of Mn2+, irrespective of which template-primer is used to direct the DNA synthesis. The action of pyridoxal phosphate on the substrate binding site may be deduced from the observations that: (a) only the substrate triphosphate is able to reverse the pyridoxal phosphate-mediated inhibition; (b) the inhibition kinetics exhibit a classical competitive pattern with the substrate; (c) analogous to substrate deoxynucleoside triphosphates, the inhibitor is also accepted only in the form of its divalent metal ion complex; and (d) substrate site-specific labeling of RLV DNA polymerase has been shown to occur by linking covalently the pyridoxal phosphate bound to a lysine residue at the substrate binding site.
The interaction of the antibiotic netropsin with calf thymus DNA, T4 DNA and poly(dA-dT) · poly(dA-dT) in complexes with sequential polypeptides containing repetitive lysine sequences and histone H1 was investigated using circular dichroism spectroscopy and equilibrium dialysis. Both soluble DNA-polypeptide complexes and insoluble complexes showed binding of netropsin. The possibility of displacement of polypeptides from DNA binding sites by competition with netropsin molecules was eliminated by experiments using 14C-labelled polypeptides. From the analysis of CD titration behavior as well as from the results of equilibrium dialysis studies it follows that netropsin does not compete with polypeptides for DNA binding sites, which suggests that these two ligands occupy different sites. Various explanations for minor differences in the CD behavior of the bound netropsin in the saturation region are also discussed.
The arrangement of the globin structural genes has been examined in murine erythroleukemia cells, strain DS19, and several related inducer-resistant variant cell lines. One fragment larger than 20 kilobases and six globin gene-containing fragments between 10 and 1.9 kilobases in size are detected in EcoRI-cleaved purified DNA prepared from strain DS19. By comparison, when isolated nuclei are digested with EcoRI, only two globin gene-containing fragments are detected, one greater than 20 kilobases and the other 1.9 kilobases. Of seven cell lines derived from DS19 and resistant to inducers, six had similar patterns to DS19 of globin gene-containing EcoRI-generated DNA fragments from nuclei and from purified DNA. One cell line, DR10, a DMSO-resistant cell line, lacks the 1.9 kilobase fragment after digestion of either nuclei or purified DNA. The 1.9 kilobase fragment hybridizes with α-globin cDNA but not with the β-globin cDNA, suggesting either rearrangement or deletion of an α-globin gene-like fragment in DR10 DNA.
The banding of ribonucleoproteins in metrizamide has been characterised using yeast ribosomes as a model system. Metrizamide does not dissociate ribosomes but it can facilitate the loss of loosely bound proteins. The buoyant density of fixed ribosomes in metrizamide gradients increases dramatically in the presence of low concentrations of Mg2+, whilst high Mg2+ concentrations give rise to multiple bands of higher density. These phenomena can be explained in terms of the binding of Mg2+ to high and low affinity sites as proposed for Escherichia coli ribosomes.
Ribosomal 5 S RNA synthesis in Chinese hamster V79 cells treated with an inhibitory antibiotic of protein synthesis, cycloheximide, was quantitated by hybridization of RNA preparations with plasmid ColE1-pSC101 DNA carrying Xenopus 5 S DNA. In V79 cells, cycloheximide produced a notable decrease in the production of the higher molecular weight ribosomal RNA (rRNA) fraction, whereas the accumulation of both 5 S RNA and polyadenylate-containing messenger RNA was much less affected. When the cellular protein synthesis was inhibited by over 85% of the control, accumulation of 5 S RNA and of rRNA was respectively 40-50% and about 10% of the control. The size distribution analysis of RNA species revealed that 5 S RNAs obtained from V79 cells after treatment with 0.1 microgram/ml or 2.0 microgram/ml cycloheximide were indistinguishable from control. Inhibition of precursor uptake into 5 S RNA by cycloheximide was apparently phase-specific during the cell cycle and was 44-67% and 78-85% of the control for the S and the M phase, respectively. The responses of RNA polymerase III activities to cycloheximide in isolated nuclei from each phase of the cell cycle correlated very well with those of precursor uptake into 5 S RNA observed in vivo, while total solubilized RNA polymerase activities showed no inhibition by the drug at any phase of the cell cycle. Cytosine arabinoside, a specific inhibitor of DNA synthesis, did not cause any decrease in the cellular level of 5 S RNA.
When added to the culture medium of thyroid cells isolated from diffuse nontoxic goiter, thyrotropin increased the poly(adenylic acid) content and the template activity of the unfractionated RNA. This increase was correlated with higher thyroglobulin messenger activity, as demonstrated by specific immunoprecipitation of the labeled peptides synthesized in two heterologous cell-free systems. When RNAs were separated in a sucrose gradient, thyrotropin was shown to enhance the poly(adenylic acid) content and template activity of fractions with sedimentation coefficients of 34, 23 and 15 S. Specific immunoprecipitation showed that a thyroglobulin messenger activity was present in these three fractions. Another way by which thyrotropin regulates the thyroid protein synthesis is suggested by the shift of poly(adenylic acid)-containing RNA to large polysomes when thyroid cells were cultured in the presence of the hormone.
Native 40 S particles from Krebs II mouse ascites tumor cells were isolated on a large scale. A nonribosomal protein moiety of about 30 proteins could be removed from the ribosomal particles by treatment with 250 mM KCl. These proteins were analysed by two-dimensional polyacrylamide gel electrophoresis and turned out to be mostly acidic in nature. The molecular weights of about 17 proteins were determined by three-dimensional gel electrophoresis. Radioactively labelled nonribosomal protein spots were excised from two-dimensional gel electrophoresis. Radioactively labelled nonribosomal protein spots were excised from two-dimensional gels and transferred directly or after electrodialysis onto the third dimension gel. The proteins fell into a molecular weight range from about 20,000 to 300,000.
After a nutritional shift from a protein-free diet to a diet containing 50% casein, the activity of DNA ligase increases in intact rat liver in correlation with the induction of hepatic DNA replication. The treated rat liver as well as control rat liver contains a single species of DNA ligase having a sedimentation coefficient of about 5.5 S. The administration of cycloheximide in vivo completely inhibits the increase in DNA ligase activity and in DNA synthesis, indicating that DNA ligase is induced in the hepatic cells replicating DNA. In contrast to DNA ligase, DNA kinase is unchanged in the activity level by the dietary manipulation.
Nuclear triiodothyronine-binding proteins (NTBP) which are at present considered as a nuclear receptor for triiodothyronine (T3) have been partially purified (about 100-fold) from rat liver nuclei by Sephadex G-100 gel filtration and DEAE-Sephadex chromatography and incubated with [125I]-T3([125I]T3-NTBP complexes). Their ability to bind to DNA and chromatin was analyzed by using in the first case rat liver or calf thymus DNA either soluble or coupled to Sepharose 4B and in the second case expanded chromatin preparations from residual chromatin after NTBP extraction (extracted chromatin) or from whole nuclei (total chromatin). [125I]T3-NTBP complexes could be rapidly and totally bound to DNA; chromatin binding was slower and less efficient on a DNA weight basis, particularly with total chromatin. In both cases, binding presented similar characteristics: it was highly sensitive to concentration of KCl and divalent cations; it depended upon the presence of reducing agents and probably the maintenance of a proper conformation of the receptor molecule; it did not need the presence of bound hormone; it was inhibited by ethidium bromide, actinomycin D and heparin. DNA binding of [125I]T3-NTBP complexes was similar with eukaryotic DNAs (rat, calf), reduced with Escherichia coli DNA, synthetic poly[d(A-T)] and heat-denatured DNA, and almost non-existent with poly(dA) or yeast RNA. No saturation of rat liver DNA could be detected, suggesting a capacity higher than 130 pmol NTBP/mg DNA. Saturation curves were observed in only three experiments out of six using total chromatin preparations and suggested capacities about 50-fold higher than the physiological concentrations of NTBP in nuclei (0.5 pmol/mg DNA). Furthermore, NTBP binding was significantly lower with spleen chromatin preparations as compared to liver ones. Thus, the T3 receptor appears as a DNA binding protein; it is suggested from our in vitro chromatin binding studies that DNA could be the major locus of NTBP binding in chromatin. The extent of DNA binding in chromatin is limited by other chromatin components in a more pronounced manner with the least modified chromatin preparations and with chromatin preparations from spleen, a tissue poorly responsive to thyroid hormones. Nevertheless, another chromatin localization of the T3 receptor cannot be excluded.
The in vitro transcription of chloroplast DNA (ctDNA) is studied using a DNA-protein complex isolated from spinach plastids. The RNA products are compared to the in vivo synthesized ctRNA by competition for hybridization. At least 80% of the in vitro RNA sequences are present in vivo. Modifications of ionic strength or introduction of heparin in the reaction medium has an important effect on transcriptional activity of the complex. Furthermore, the length of the RNA chains increases ionic strength and amount of heparin. The RNA products are analysed by Southern hybridizations to EcoRI cTDNA fragments. Changes in the ionic strength or in the amount of heparin modify heterogeneously the transcription of the various DNA regions. The quantitative distribution of transcripts among the ctDNA fragments is used as evidence for the selectivity of the transcription. The activity of the DNA-protein complex is much more resistant to high ionic strength than an heterologous transcription system using Escherichia coli RNA polymerase and ctDNA. This latter system transcribes less ctDNA fragments.
Alanyl- and leucyl-tRNA synthetases from baker's yeast were purified to homogeneity in the presence of the protease inhibitor phenylmethylsulfonyl fluoride. Both consist of single polypeptide chains of 118 000 and 125 000 daltons, respectively, as determined by polyacrylamide gel electrophoresis under denaturing conditions. The monomeric structure of leucyl-tRNA synthetase differs from the dimeric one obtained previously in the absence of protease inhibitors. This illustrates the sensitivity of the synthetases to proteolytic actions and indicates that native structures can only be obtained under optimal protecting conditions. Alanyl- and leucyl-tRNA synthetases differ with respect to pH optimum (6.5 and 8.5, respectively), Michaelis constant for amino acid (1 mM and 0.03 mM, respectively) and in the rate-limiting step for the tRNA aminoacylation reaction. Whereas the catalytic step itself was rate-limiting for alanyl-tRNA synthetase, a step occurring after this was rate-limiting for leucyl-tRNA synthetase.
Exposure of streptomycin-resistant cells to puromycin results in uptake of dihydrostreptomycin comparable to that found with streptomycin-sensitive cells. This finding indicates that the enhanced phase of uptake, previously reported only in sensitive cells, may result from an increase in internal binding sites, presumably run-off ribosomes. The increased uptake of dihydrostreptomycin resulting from exposure to puromycin is greatest in both sensitive and resistant cells at concentrations below 100 microgram/ml. At 100 microgram/ml, exposure to puromycin in vivo results in significant, but not complete, polysome degradation and inhibition of protein synthesis. At 500 microgram/ml, where polysome degradation is complete in less than 2 min and where growth and protein synthesis are inhibited more than 90%, uptake of dihydrostreptomycin by both sensitive and resistant cells is inhibited. Puromycin has no effect on binding of dihydrostreptomycin to 70-S monosomes, as measured by equilibrium dialysis. The increased uptake of dihydrostreptomycin by resistant cells resulting from exposure to puromycin has no effect on viability. Addition of N-ethylmaleimide immediately and completely inhibits the puromycin-induced uptake of dihydrostreptomycin even when added after substantial polysome degradation has occurred.
The degradation of a mixture of four 5'-ribonucleotides (AMP, GMP, CMP and UMP), yeast RNA, yeast phenylalanine tRNA, and tobacco mosaic virus RNA (TMV-RNA) with ozone (concentration in inlet gas, 0.1-0.5 mg/l) was examined in a phosphate buffer (pH 6.9). In the case of the mixture, GMP alone was degraded in the initial stage. In the ozonization of yeast RNA, the guanine moiety was less vulnerable to attack by ozone than in the case of free GMP, but it again degraded most rapidly among the four nucleotides. In the treatment of tRNA with ozone, the guanine moiety degraded first. When the numbers of degraded nucleotides reached 4.8 (remaining amino acid acceptor activity was 3.6%), the polyacrylamide gel electrophoresis of the ozonized tRNA gave a single band with the same mobility as that of the intact tRNA. It is evident that ozonolysis of tRNA proceeded without cleavage of the polynucleotide chain. In the case of TMV-RNA, the loss of the infectivity by ozone proceeded rapidly within 30 min and was followed by preferential degradation of the guanine moiety. The outstanding lability of the guanine moiety observed in each case is discussed in connection with the inactivation of tRNA and TMV-RNA.
hnRNP are made of two classes of unit, monoparticles and heterogeneous complexes. The monoparticles are much more easily dissociated by salt than the heterogeneous complexes. We made use of this differential salt sensitivity to determine the localization of snRNA in hnRNP. 1, About 50% of the snRNA were released by NaCl under the conditions of dissociation of monoparticles, U1 RNA which was enriched in monoparticles was preferentially released. 2, When the proteins resistant to salt dissociation were digested with proteinase K, an additional small proportion of snRNA was released, in particular a species designated 5 Sa RNA. Therefore, 5 Sa RNA seems to be preferentially associated with the proteins of heterogeneous complexes. 3, 40% of the snRNA remained associated with the hnRNA in the absence of any detectable protein. U1 and U2 RNA were the major RNAs in this fraction. The same RNA pattern was obtained for phenol-extracted RNA. The results indicate that all snRNA species are associated with the proteins of monoparticles, with those of heterogeneous complexes and with hnRNA. The existence of these pools of snRNA may reflect different functional states.
An in vitro protein synthesizing system consisting of thylakoid-bound polysomes supplemented with an S-100 from Escherichia coli has been used to investigate chloroplast membrane protein synthesis in synchronous cultures of Chlamydomonas reinhardii. The cell-free system produces at least one thylakoid membrane protein, D-2, on the basis of electrophoretic mobility and peptide mapping. Rough thylakoid membranes isolated in the middle and late light portion of the cell cycle synthesize substantial amounts of D-2 in vitro while the synthesis of D-2 was not detected by rough thylakoids isolated early in the light period or midway through the dark portion of the cell cycle. The same result was found when the synthesis of D-2 was investigated in vivo by pulse-labeling synchronous cell cultures with [3H]arginine. This provides further evidence for the faithful synthesis of polypeptide D-2 in vitro. These results show that one function of thylakoid-bound ribosomes is to synthesize thylakoid membrane proteins. The results also show the cell cycle regulation of membrane protein synthesis that exists within the chloroplast on thylakoid-bound ribosomes.
