The effect of removal or replacement with proflavine of the Y base in the anticodon loop of yeast tRNAPhe on binding into the acceptor or donor sites of reticulocyte ribosomes

Phe-tRNA from yeast has a highly modified nucleoside, called Y, adjacent to the 3′ side of its anticodon, that can be removed or replaced with proflavine. In a protein-synthesizing system from rabbit reticulocytes, poly (U)-directed binding and polyphenylalanine synthesis are low with these modified Phe-tRNA species relative to the corresponding values with unmodified Phe-tRNA. However, polymerization can be increased with relatively large amounts of elongation factor I. The modified Phe-tRNA species bound to the ribosomes with poly(U) either in the presence or absence of elongation factor I and GTP is immediately reactive in the peptidyl transferase reaction measured by the formation of diphenylalanine or phenylalanyl-puromycin. It appears to have been bound directly into the donor ribosomal site by either the nonenzymatic mechanism involving Mg²⁺ or by the enzymatic mechanism involving EF-I and GTP.     

Identification of an altered elongation factor in temperature-sensitive mutant ts 7'-14 of Saccharomyces cerevisiae

Postpolysomal extracts from wild-type (wt A364A) and temperature-sensitive (ts 7'-14) yeast cells were preincubated for short periods of time at the nonpermissive temperature (37-41 degrees C) prior to incubations for protein synthesis at 20 degrees C. Whereas wt A364A extracts were relatively unaffected by preincubation at the elevated temperature, mutant extracts lost their ability to translate exogenous natural mRNA and poly(U). Phe-tRNA synthetase and ribosomes from ts 7'-14 cells were not inactivated by preincubation at 37-41 degrees C, but a cytosolic component required for chain elongation, as measured by poly(U) translation, was extensively inactivated. The three elongation factors (EF-1, EF-2, and EF-3) required for chain elongation in yeast were resolved chromatographically. Only one factor, EF-3, was able to restore the poly(U)-translational activity of mutant extracts inactivated at the elevated temperature. Heat-inactivated yeast cytosols, which did not support protein synthesis with yeast ribosomes, were perfectly able to translate poly(U) with rat liver ribosomes, which require only EF-1 and EF-2. These and other experiments indicated that the genetically altered component in 7'-14 mutant cells is EF-3.     

Isolation of ribosomal subparticles from human placenta containing intact rRNA and determination of the functional activity of the 80S ribosome

The method for isolation of human placenta ribosomal subunits containing intact rRNA has been determined. The method uses fresh unfrozen placenta. Activity of 80S ribosomes obtained via reassociation of 40S and 60S subunits in non-enzymatic poly(U)-mediated Phe-tRNAPhe binding, was near 75% (maximal [14C]Phe-tRNA(Phe) binding was 1.5 mol Phe-tRNA(Phe) per mol of 80S ribosomes). Activity of 80S ribosomes with damaged rRNA isolated from frozen placenta was 2 times lower (the maximum level of poly(U)-dependent Phe-tRNA(Phe) binding was 0.7 mol per mol of ribosomes). The activity 80S ribosomes in poly(U)-mediated synthesis of polyphenylalanine was determined by using fractionated ("ribosomeless") protein synthesising system from rabbit reticulocytes. In this system up to the 50 mol of Phe residues per mol of 80S ribosomes are incorporated in acid insoluble fraction in 1 hour, at 37 degrees C. The obtained level of [14C]phenylalanine incorporation is three times as much as the amount of Phe residues observed for the ribosomal subunits, isolated from frozen placenta.     

Demonstration of an endogenous, competitive inhibitor(s) of [3H] diazepam binding in bovine brain.

An endogenous inhibitor(s) of [³H] diazepam binding to synaptosomes has been demonstrated in bovine brain. The inhibitory activity of crude extracts is heat stable, dialyzable, and not affected by ether extraction. Three distinct peaks of inhibitory activity were resolved using Sephadex G-25 chromatography. The lowest molecular weight peak (<700 daltons) had the highest specific inhibitory activity and its inhibition of [³H] diazepam binding was competitive. A similar low molecular weight fraction was not observed in either muscle or liver suggesting that it may be unique to brain. Thin layer chromatography of the Sephadex G-25 fractions revealed a discrete band of inhibitory activity in the two low molecular weight peaks.     

Isolation of ribosomal subunits containing intact rRNA from human placenta: estimation of functional activity of 80S ribosomes.

We have elaborated a method for the isolation of ribosomal subunits from fresh unfrozen human placenta containing intact rRNA and a complete set of ribosomal proteins. Activity of 80S ribosomes obtained by reassociation of 40S and 60S subunits in nonenzymatic poly(U)-dependent binding of Phe-tRNA(Phe) was equal to 80% (above 1.5 mol [14C]Phe-tRNA(Phe) is coupled to 1 mol of ribosomes). The activity of 80S ribosomes in poly(U)-directed synthesis of polyphenylalanine was tested in a polysome-free protein-synthesizing system from rabbit reticulocytes. About 100 mol of phenylalanine residue was polymerized by a mole of ribosomes at a rate of 0.83 residues per minute in this system (2 h, 37 degrees C).     

Aminoacyl-tRNA-elongation factor Tu-ribosome interaction leading to hydrolysis of guanosine 5'-triphosphate

We investigated the elongation factor Tu (EF-Tu) dependent binding of Phe-tRNA and Phe-tRNAs with the nicks at positions 46, 37, and 17 to the Escherichia coli 70S ribosome-poly(U)-tRNAPhe complex. Binding of Phe-tRNA1-45 + 47-76, Phe-tRNA1-36 + 38-76, or Phe-tRNA1-16 + 17-76 to the 70S ribosome has been found to be poly(U) X tRNA dependent and, similar to that of intact Phe-tRNA, is inhibited by the antibiotic thiostrepton. We have further found that, contrary to a previous report [Modolell, J., Cabrer, B., Parmeggiani, A., & Vazquez, D. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 1796], the EF-Tu-ribosome GTPase mediated by Phe-tRNA is not inhibited by thiostrepton; rather, the drug stimulates the endogenous GTPase of the EF-Tu X 70S ribosome. Phe-tRNA fragments 47-76, 38-76, and 17-76 all promote the EF-Tu X GTPase reaction in the presence of 70S ribosome-poly(U)-tRNAPhe yeast. Moreover, since the GTPase-promoting activities of both the short and long fragments are similar, it appears that the most important aminoacyl transfer ribonucleic acid (aa-tRNA) interaction with EF-Tu occurs alongside its 3' quarter. Thiostrepton slightly stimulates the GTPase activity of these Phe-tRNA fragments. Although the Phe-tRNA1-36 + 38-76 cannot bind to poly(U) during its binding to 70S ribosomes, its binding at high Mg2+ concentration occurs at the A site. Thus, most of the bound modified Phe-tRNA functions as the acceptor in the peptidyltransferase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Codon-specific interaction of uncharged transfer-RNA with eukaryotic ribosomes.

Rat liver ribosomes bound [32P]tRNAPhe in both a codon-dependent and codon-independent manner. The codon-dependent binding was studied further by utilising the ability of the unchanged tRNAPhe to inhibit the poly(U)-directed binding of [3H]Phe-tRNA to ribosomes. At least part of the codon-dependent binding of uncharged tRNA appears to be to the ribosomal A-site.     

Regulation of poly(A) polymerase activity and poly(A)+ RNA in germinated wheat embryos under conditions of pathogenesis

The induction of poly(A) polymerase was accompanied by a rise in the level of poly(A)⁺ RNA during early germination of excised wheat embryos (48 h). Fractionation of this RNA-processing enzyme by acrylamide gel electrophoresis and also by molecular sieving on Sephadex G-200 revealed a single molecular form of poly(A) polymerase with a molecular weight of 125 000. Wheat poly(A) polymerase specifically catalyzed the incorporation of [³H]AMP from [³H]ATP into the polyadenylate product only in the presence of primer RNA. Substitution of [³H]ATP by other labelled nucleoside triphosphates, such as [³H]GTP, [³H]UTP or [α-³²P]CTP in the assay mixture did not yield any labelled polynucleotide reaction product. The ³H-labelled reaction product was retained on poly(U)-cellulose affinity column and was not degraded by RNAase A and RNAase T₁ treatment. In addition, the nearest-neighbour frequency analysis of the ³²P-labelled reaction product predominantly yielded [³²P]AMP. Thus, characterization of the reaction product clearly indicated its polyadenylate nature. The average chain length of the [³H]poly(A) product was 26 nucleotides. Infection of germinating wheat embryos by a fungal pathogen (Drechslera sorokiana) brought about a severe inhibition (62–79%) of poly(A) polymerase activity. Concurrently, there was a parallel decrease (73%) in the level of poly(A)⁺ RNA. Inhibition of poly(A) polymerase activity in infected embryos could be due to enzyme inactivation, which in turn brought about a downward shift in the level of poly(A)⁺ RNA. The crude extract of the cultured pathogen contains a non-dialysable, heat-labile factor, which, along with a ligand, inactivates (65–74%) poly(A) polymerase in vitro. The fungal extracts also contained a dialysable, heat-stable stimulatory effector which activated wheat poly(A) polymerase (3.6–4.0-fold stimulation) in vitro. However, the stimulatory fungal effector was not expressed in vivo, but was detectable after the inhibitory fungal factor had been destroyed by heat-treatment in our in vitro experiments.     

The effect of an antiviral peptide on the ribosomal reactions of the peptide elongation enzymes, EF-I and EF-II

A basic peptide with antiviral properties isolated from pokeweed is shown to inhibit the synthesis of globin and phenylalanine peptides on ribosomes isolated from rabbit reticulocytes. The inhibition appears to involve a specific effect of the peptide inhibitor on the larger ribosomal subunit that can be produced at a ratio of inhibitor to ribosomes of less than one to one. Ribosomes treated with the inhibitor have a reduced capacity to support enzymatic binding of Phe-tRNA to ribosomes and GTP hydrolysis caused by the elongation enzyme, EF-I. Treated ribosomes exhibit a concomitant capacity for increased GTP hydrolysis by EF-II but do not efficiently support EF-II-dependent binding of [³H]GTP. Such binding appears to involve the formation of an EF-II·GDP·ribosome complex. Thus, the inhibitor has an effect on GTP-dependent reaction carried out by both of the peptide elongation enzymes. The relation between these effects in the reticulocyte system is discussed in relation to the effects of siomycin or thiostrepton in blocking GTP hydrolysis by EF-T and EF-G on prokaryotic ribosomes.     

Studies of odd bases in yeast mitochondrial tRNA: Absence of the fluorescent “Y” base in mitochondrial DNA coded tRNAPhe, absence of 4-thiouridine

Reversed phase chromatography of mitochondrial [³H] Phe-tRNA from $\text{Saccharomyces cerevisiae}$ shows only one peak which elutes distinctly from cytoplasmic [¹⁴C] Phe-tRNA. Mitochondrial tRNA^Phe from this peak hybridizes specifically with ?⁺ and a ?^? mitochondrial DNA. Search for rare bases in mitochondrial tRNA shows the absence of the eukaryotic “Y” base and of the prokariotic s⁴U base.     

Endonucleolytic cleavage of murine leukemia virus 35S RNA by microsome-associated nuclease.

Moloney murine leukemia virus 35S RNA (molecular weight 3 to 3.4 × 10⁶) is cleaved by nuclease activity present in microsomal fractions from MLV infected or uninfected mouse embryo cells to two RNA species of approximate molecular weights 1.8 × 10⁶ and 1.5 × 10⁶. Microsomal fractions from MLV infected and uninfected cells also contained nucleolytic activity that solubilized [³H]poly(A)·poly(U) but not [³H]poly(C) or [³H]poly(U); the cleavage of poly(A)·poly(U) was inhibited by ethidium bromide. The cleavage of MLV RNA was also inhibited by ethidium bromide, suggesting double stranded regions in 35S RNA as the site of cleavage.     

Differential accumulation and localization of maternal poly(A)-containing RNA during early development of the ascidian,Styela

The regional distribution of poly(A)⁺ RNA was examined in sections of Styela oocytes and fertilized eggs by in situ hybridization with [³H]poly(U). The nucleus and cytoplasm of previtellogenic oocytes contain equivalent densities of [³H]poly(U) binding sites. The concentration of these sites is reduced in the cytoplasm, but not the nucleus, during vitellogenesis. Consequently, the germinal vesicle (GV) plasm of mature oocytes is characterized by an eightfold elevation in [³H]poly(U) binding activity relative to the surrounding cytoplasm. The distinctive cytoplasmic regions of the mature oocyte do not exhibit differential concentrations of [³H]poly(U) binding sites. Following fertilization which triggers GV breakdown, meiosis, and ooplasmic segregation, the high density of [³H]poly(U) binding sites characteristic of the GV plasm is conserved in the basophilic cytoplasm during its extensive migration and eventual accumulation in the animal hemisphere of the egg. The insensitivity of the [³H]poly(U) binding sites of the basophilic cytoplasm to actinomycin D suggests that they are of maternal origin. It is concluded that maternal poly(A)⁺ RNA is subject to differential accumulation in the GV plasm and its derivative ooplasm during the early development of Styela.     

Evidence for in vivo compartmentation of phenylalanyl-tRNA ligase in amphibian oocytes

The in vivo activity of phenylalanyl-tRNA ligase of Xenopus laevis oocytes was assayed by measuring the esterification of microinjected yeast tRNA^Phe with [¹⁴C]phenylalanine added to the extracellular medium. The three enzyme substrates, ATP, phenylalanine, and tRNA^Phe, are present in the in vivo assay at saturating concentrations as seen by the fact that microinjection into the cell of additional amounts of these compounds does not increase the quantity of [¹⁴C]Phe-tRNA^Phe formed. The in vivo activity of Phe-tRNA ligase in oocytes at several stages of development is less than 10% of the in vitro activity measured in homogenates of the same cells. The in vivo assay of Phe-tRNA ligase in oocytes that have been microinjected with this enzyme partially purified from X. laevis ovary shows that the enzyme is not inhibited by the cellular conditions. The conclusion drawn from these experiments is that a large fraction of the Phe-tRNA ligase present in oocytes is in a cellular compartment which is not available to the injected tRNA.     

Density-dependent inhibition of growth: Inhibitorydiffusible factors from 3T3- and rous sarcoma virus (RSV)-transformed 3T3 cells

We recently fractionated, from the culture medium of 3T3 cells, a thermolabile inhibitory diffusible factor (IDF_N) with a molecular weight of about 40,000 daltons, which decreased nucleic acids synthesis of stimulated target 3T3 cells. In the present publication the inhibitory activities of IDF_N (produced by 3T3 cells) and IDF_T (produced by RSV-transformed 3T3 [3T3 SRA/H] cells) on 3T3 and 3T3 SRA/H cells have been compared. The inhibitory activity of IDF_N decreased (by a mean of 57%) when it was tested on transformed instead of 3T3 cells. On the other hand IDF_T was able to decrease ¹⁴C-inosine incorporation in target 3T3 cells. However, the inhibitory activity of IDF_T decreased (by mean 50%) when tested on 3T3 SRA/H instead of 3T3 cells. Therefore, transformed cells produced an inhibitory factor but were less sensitive than 3T3 cells to its inhibitory activity. The inhibitory activity of IDF_T on 3T3 SRA/H cells was only 20% of the inhibitory activity of IDF_N on 3T3 cells. This appreciable difference is of particular interest, since it could explain the release of density-dependent inhibition of growth (DDI) in transformed 3T3 SRA/H cells. Furthermore, it provides more evidence for the hypothesis that, in 3T3 cells, DDI of growth is due to the release of an inhibitory molecule into the medium, and that IDF_N is in fact, the inhibitory molecule involved in this phenomenon.     

Stimulation of enzymatic phe-tRNA binding to mammalian ribosomes by thallium ions at concentrations blocking other ribosomal functions.

Thallium acetate (TIOAc) effectively stimulates poly(U)-directed Phe-tRNA binding to mouse ascitic tumour ribosomes under conditions when other ribosomal functions are completely blocked. The TI+ optimum is about 200 mM. The reaction is stimulated by EF-1, but not significantly by GTP. EF-1-dependent ribosomal GTPase is inhibited by T1+. The isolated Phe-tRNA . ribosome complex is relatively stable. The bound Phe-tRNA does not react with puromycin in the presence of 175 mM KCl. The complex formed in the presence of 90-100 mM TlOAc can, after isolation, be directly utilized for polyphenylalanine synthesis. The complex formed at 200 mM TlOAc is less active, apparently because of damage to the 60-S subunits. TlOAc at low concentrations (8 mM) stimulates K+ -containing poly(U)-translating systems, probably by stabilizing the translation complex.     

Asparagine synthesis in pea leaves, and the occurrence of an asparagine synthetase inhibitor

Asparagine is present in the mature leaves of young pea (Pisum sativum cv Little Marvel) seedlings, and is synthesized in detached shoots. This accumulation and synthesis is greatly enhanced by darkening. In detached control shoots, [¹⁴C]aspartate was metabolized predominantly to organic acids and, as other workers have shown, there was little labeling of asparagine (after 5 hours, 3.1% of metabolized label). Addition of the aminotransferase inhibitor aminooxyacetate decreased the flow of aspartate carbon to organic acids and enhanced (about 3-fold) the labeling of asparagine. The same treatment applied to darkened shoots resulted in a substantial conversion of [¹⁴C]aspartate to asparagine, over 10-fold greater than in control shoots (66% of metabolized label), suggesting that aspartate is the normal precursor of asparagine.

Only traces of glutamine-dependent asparagine synthetase activity could be detected in pea leaf or root extracts; activity was not enhanced by sulfhydryl reagents, oxidizing conditions, or protease inhibitors. Asparagine synthetase is readily extracted from lupin cotyledons, but yield was greatly reduced by extraction in the presence of pea leaf tissue; pea leaf homogenates contained an inhibitor which produced over 95% inhibition of an asparagine synthetase preparation from lupin cotyledons. The inhibitor was heat stable, with a low molecular weight. Presence of an inhibitor may prevent detection of asparagine synthetase in pea extracts and in Asparagus, where a cyanide-dependent pathway has been proposed to account for asparagine synthesis: an inhibitor with similar properties was present in Asparagus shoot tissue.

     

Comparison of the modes of action of a Vero toxin (a Shiga-like toxin) from Escherichia coli, of ricin, and of alpha-sarcin.

The modes of action of a Vero toxin (VT2 or Shiga-like toxin II) from Escherichia coli, of ricin, and of alpha-sarcin were compared. Elongation factor 1 (EF1) and GTP-dependent Phe-tRNA binding to ribosomes in the presence of poly(U) was inhibited by these three toxins, but EF1 and guanylyl (beta, gamma-methylene)-diphosphate-dependent Phe-tRNA binding was inhibited by alpha-sarcin only. EF1- and Phe-tRNA-dependent GTPase activity was inhibited by these toxins, but nonenzymatic binding of Phe-tRNA was not. The turnover rate of EF1 binding to ribosomes during Phe-tRNA binding was also decreased by these three toxins. The addition of EF1 recovered the inhibition of Phe-tRNA binding to ribosomes by VT2 and ricin but not by alpha-sarcin. The formation of and EF2- and GTP-dependent puromycin derivative of phenylalanine was inhibited slightly by the three toxins, indicating that translocation is not influenced significantly by them. EF2-dependent GTPase activity was stimulated by these toxins, and especially by VT2 and ricin. In contrast, the binding of EF2 to ribosomes was inhibited strongly by VT2 and ricin, and slightly by alpha-sarcin. The stimulation of EF2-dependent GTPase activity by the toxins may compensate for the decrease of EF2 binding to ribosomes which they caused during translocation. In total, these results indicate that VT2 and ricin inhibit protein synthesis through the disturbance of the turnover of EF1 binding to ribosomes during aminoacyl-tRNA binding to ribosomes, and that alpha-sarcin inhibits the synthesis through the inhibition of the binding of the complex of Phe-tRNA, EF1, and GTP to ribosomes.     

Microspectrophotometric determination of the relative contents of nucleic acids in Pinus silvestris pollen

Based on inhibitor studies it has been concluded that in photoregulated processes of plant development, light induces differential gene expression. Using affinity chromatography of double labelled polysomal RNA on poly(U)-sepharose 4B, we were able to demonstrate different ³H/¹⁴C ratios for the bound poly(A) containing fraction (mRNA) when compared with the unbound fraction: when [³H]uridine was present in the light induced sample and [¹⁴C]uridine in the dark control, in the bound material the ³H/¹⁴C ratio was found to be higher than in the unbound fraction and vice versa. No such shift was observed, when both sample and control were kept in the dark. Our data are interpreted to provide evidence for photoinduced de novo synthesis of mRNA.Abbreviations CHI cycloheximide - CTAB N-acetyl-N,N,N-trimethylammoniumbromide - poly(A) poly-adenylic acid - Butyl-PBD 2-(4-t-Butylphenyl)-5-(4-biphenylyl)-1,3,4-oxadiazole