Polyuridylic Acid-directed Polyphenylalanine Synthesis Research Articles

PpGpp inhibits peptide elongation cycle of chloroplast translation system in vitro.

Chloroplasts possess common biosynthetic pathways for generating guanosine 3',5'-(bis)pyrophosphate (ppGpp) from GDP and ATP by RelA-SpoT homolog enzymes. To date, several hypothetical targets of ppGpp in chloroplasts have been suggested, but they remain largely unverified. In this study, we have investigated effects of ppGpp on translation apparatus in chloroplasts by developing in vitro protein synthesis system based on an extract of chloroplasts isolated from pea (Pisum sativum). The chloroplast extracts showed stable protein synthesis activity in vitro, and the activity was sensitive to various types of antibiotics. We have demonstrated that ppGpp inhibits the activity of chloroplast translation in dose-effective manner, as does the toxic nonhydrolyzable GTP analog guanosine 5'-(β,γ-imido)triphosphate (GDPNP). We further examined polyuridylic acid-directed polyphenylalanine synthesis as a measure of peptide elongation activity in the pea chloroplast extract. Both ppGpp and GDPNP as well as antibiotics, fusidic acid and thiostrepton, inhibited the peptide elongation cycle of the translation system, but GDP in the similar range of the tested ppGpp concentration did not affect the activity. Our results thus show that ppGpp directly affect the translation system of chloroplasts, as they do that of bacteria. We suggest that the role of the ppGpp signaling system in translation in bacteria is conserved in the translation system of chloroplasts.

Effects and molecular action of ribosome-inactivating proteins on ribosomes from Streptomyces lividans

The effects of 29 type 1 and 2 type 2 ribosome-inactivating proteins (RIPS) from plants on polyuridylic acid-directed polyphenylalanine synthesis carried out by purified ribosomes from Streptomyces lividans were studied. Only dianthin 32, saporins R1 and R3, momordin I, trichokirin, Hura crepitans RIP 5 from latex, crotins 2 and 3, and PAPs C, R, and S, inhibited polyphenylalanine synthesis. Both the type 2 RIPs ricin and volkensin were ineffective on translation. The magnesium concentration affected the inhibition of translation to a considerable extent. Upon treatment with inhibitory RIPs, extraction of rRNA and further treatment with acid aniline, S. lividans ribosomes released an RNA fragment of about 130 nucleotides. The 5′ terminal sequence of this rRNA fragment was 5′-GAGGACCGGGACGGACGAACCUCUGGUGUGCCAGUUGU-3′, similar to the sequence obtained in Escherichia coh. This indicates that the most probable molecular action of these RIPs on S. lividans and E. coli ribosomes is the same: depurination of the rRNA at a site relevant to the translation mechanism and that has been highly conserved throughout evolution.

Characterization of mesosomes of Micrococcus luteus: isolation and properties of mesosomal ribosomes, and localization of penicillin-binding proteins in mesosomal membranes.

Mesosomes were isolated and purified from Micrococcus luteus under hypertonic conditions throughout preparation processes. The purified mesosomal preparation was composed of closed tubules and vesicles. Electron-dense ribosome-like particles were observed within the isolated mesosomal vesicles by electron microscopy. The ribosome-like particles were isolated from the purified mesosomes by a procedure involving solubilization of the membranes with detergents followed by centrifugation on a linear density gradient of sucrose. The isolated particles have a sedimentation coefficient of 70S in the presence of 10 mM Mg2+, when Mg2+ concentration was lowered to 0.1 mM, the particles were dissociated into two sub-particles of 30S and 50S. The 70S particles had the same appearance as cytoplasmic 70S ribosome particles upon observations of negatively stained preparations. These findings indicate that mesosomal tubules contain ribosomes. The isolated mesosomal ribosomes had the ability for protein synthesis when polyuridylic acid-directed polyphenylalanine synthesis was assayed. The sensitivity of mesosomal ribosomes to inhibitors, chloramphenicol and streptomycin, for protein synthesis was significantly lower than that of both cytoplasmic and cytoplasmic membrane-bound ribosomes. In addition, three penicillin-binding proteins were detected in the mesosomal membranes. One of these was localized predominantly in the mesosomal membranes and the other two were distributed almost equally in both mesosomal and cytoplasmic membranes.

The selective release of one of the two L7/L12 dimers from the Escherichia coli ribosome induced by a monoclonal antibody to the NH2-terminal region.

Two monoclonal antibodies against different epitopes in Escherichia coli ribosomal protein L7/L12 were prepared and characterized as reported previously (Sommer, A., Etchison, J.R., Gavino, G., Zecherle, N., Casiano, C., and Traud, R.R. (1985) J. Biol. Chem. 260, 6522-6527). Both antibodies strongly inhibited polyuridylic acid-directed polyphenylalanine synthesis, ribosome-dependent GTPase activity, and the binding of elongation factor G to the ribosome at mole ratios over ribosomes of 4:1 or less. One epitope was shown to be within residues 1-73 (Ab 1-73) and the other within 74-120 (Ab 74-120). Incubation of 50 S ribosomal subunits or 70 S ribosomes with Ab 1-73, but not with Ab 74-120, leads to a partial loss of L7/L12 from the particle with no loss of any other protein. The experiment was repeated with ribosomes reconstituted with pure radioactive L7/L12 of determined specific activity in order to quantify the L7/L12 in the antibody-treated particle. The protein-deficient core particles isolated by sucrose gradient centrifugation after incubation with Ab 1-73 were found to contain, on average, two copies of L7/L12 and one Ab 1-73. The constancy of this stoichiometry in many experiments and the demonstration of Ab 1-73 on all particles indicate the presence of a homogeneous population of ribosomes, each with only one of the two L7/L12 dimers originally present. The results show a difference in the interactions of the two dimers with the ribosome and present a means of preparing ribosomes with one dimer in a specific binding site. The accompanying paper (Olson, H.M., Sommer, A., Tewari, D. S., Traut, R.R., and Glitz, D.G. (1986) J. Biol. Chem. 261, 6924-6932) shows by immune electron microscopy the location of the two antibody-binding sites and the effect of Ab 1-73 on structure.

Preparation and characterization of two monoclonal antibodies against different epitopes in Escherichia coli ribosomal protein L7/L12.

Two monoclonal antibodies with specificities for Escherichia coli 50 S ribosomal subunit protein L7/L12 were isolated. The antibodies and Fab fragments thereof were purified by affinity chromatography using solid-phase coupled L7/L12 protein as the immunoadsorbent. The two antibodies were shown to recognize different epitopes; one in the N-terminal and the other in the C-terminal domain of protein L7/L12. Both intact antibodies strongly inhibited polyuridylic acid-directed polyphenylalanine synthesis, ribosome-dependent GTPase activity, and the binding of elongation factor EF-G to the ribosome. Ratios of antibody to ribosome of 4:1 or less were effective in inhibiting these activities. Neither antibody prevented the association of ribosomal subunits to form 70 S ribosomes. The Fab fragments showed similar effects.

Mechanism of protection of protein synthesis against streptomycin inhibition in a producing strain.

The influence of streptomycin (SM) on protein synthesis in a SM-producing strain was investigated using polyuridylic acid-directed polyphenylalanine synthesis in cell-free extracts. Tolerance of protein synthesis to SM developed with increasing culture age of cells and could be attributed to a decrease in affinity of the ribosomes for SM and in increase in SM 6-kinase activity in cells. SM 6-phosphate produced from SM by SM 6-kinase did not bind to ribosomes and, furthermore, ribosome-bound SM was effectively released on phosphorylation with SM 6-kinase. Also a decrease in cell permeability to SM during the production phase may contribute in protecting protein synthesis from the antibiotic.

Changes in brain protein synthesis during the life span of male Fischer rats.

The cell-free protein synthetic activity of the postmitochondrial supernatant isolated from whole brain of 6- to 32-month-old male Fischer F344 rats was compared. Protein synthesis decreased 56% from 6 to 32 months of age. The decrease in cell-free protein synthesis was not due to an age-related increase in RNase activity. Although monomeric ribosomes (ribosomes stripped of mRNA) isolated from the brains of older rats were less active in polyuridylic acid directed polyphenylalanine synthesis, the fidelity of polyuridylic acid translation by monomeric ribosomes did not decrease with increasing age.

Comparison of ribosomes from Coxiella burnetii and Escherichia coli by gel electrophoresis, protein synthesis, and immunological techniques

Ribosomes and postribiosomal supernatant fluid (S-100) were isolated from Coxiella burnetii. The ribosomes functioned in polyuridylic acid-directed polyphenylalanine synthesis in the presence of S-100 from either C. burnetii or Escherichia coli. C. burnetii S-100 promoted translation with E. coli ribosomes. Antisera against E. coli elongation factor G and ribosomal proteins L7/L12 cross-reacted with rickettsial S-100 and ribosomes, respectively. Ribosomal proteins were analyzed by two-dimensional gel electrophoresis.

Protein synthesis in a cell-free system from an extreme thermophile. Effects of preincubation in the cold on polyuridylic acid-dependent polyphenylalanine synthesis at high temperature.

1. It was found that preincubation of the reaction mixture in the cold enhanced polyuridylic acid-directed polyphenylalanine synthesis by a cell-free extract of Thermus thermophilus HB8 at high temperature. 2. The effect of preincubation was most marked at 10-25 degrees in the presence of 20 mM Mg2+. Preincubation at 65 degrees failed to stimulate the incorporation. 3. The presence of phenylalanyl-tRNA, polyuridylic acid, and ribosomes was essential for preincubation in the cold to be effective. 4. A ternary complex of amino acyl-tRNA, polyuridylic acid, and a ribosome formed at low temperature was isolated by CPG-10 column chromatography; the isolated complex initiated polyphenylalanine synthesis effectively at high temperature. 5. The amount of the ternary complex formed depends on the preincubation time and the concentration of Mg2+. Since the amount of the complex correlated positively to the rate of polyphenylalanine synthesis at high temperature, the effectiveness of preincubation in the cold is presumably due to the formation of the ternary complex of phenylalanyl-tRNA, polyuridylic acid, and a ribosome.

Demonstration of thyroxine-stimulated incorporation of amino acid into peptide linkage in mitochondria-free system.

The observation that thyroxine stimulated in vitro protein synthesis in the absence of mitochondria (Carter, W.J., Faas, F.H., and Wynn, J (1971) J. Biol. Chem. 246, 4973-4977) has been disputed on the basis that radioactivity incorporated into protein did not represent peptide synthesis but incorporation of labeled contaminants present in the L-(U-14C) valine precursor (Sokoloff, L., and Roberts, P.A. (1972 Fed. Proc. 31, 1525). The question of mitochondrial requirement is important in determining whether thyroxine has a direct action on the polysome or causes the release of stimulatory factors from mitochondria. In this paper, thyroxine stimulation of peptide synthesis in mitochondria-free systems has been confirmed. Peptide synthesis is required for the thyroxine effect since it is dependent on the presence of polysomes and an energy source in the reaction mixture and is abolished by puromycin. The thyroxine effect is not due to incorporation of labeled contaminants since hydrolysis of labeled protein recovered from control and thyroxine-treated reaction mixtures yields the labeled amino acid precursor as the only radioactive product. Thyroxine stimulates polyuridylic acid-directed polyphenylalanine synthesis, providing further evidence that thyroxine is stimulating peptide synthesis rather than incorporation of radioactive contaminants by mechanisms other than peptide synthesis. Although thyroxine stimulates polyphenylalanine synthesis, it does not influence polyuridylic acid hydrolysis measured in the same reaction. Therefore, thyroxine stimulation of peptide synthesis is not due to prevention of hydrolysis of nucleic acid components of the reaction mixture. Thyroxine does not influence the size or specific activity of the free valine pool in the reaction mixture, indicating that observed increases in valine incorporation reflect increased peptide synthesis rather than increased specific activity of the valine precursor. The fact that thyroxine stimulates peptide synthesis using (14C)aminoacyl-tRNA precursors strengthens this conclusion. Therefore, thyroxine stimulation of protein labeling is dependent on the presence of peptide synthesis and cannot be explained by incorporation of labeled contaminants, prevention of RNA hydrolysis, or change in the specific activity of the amino acid precursor. Thyroxine causes a genuine increase in peptide synthesis by a direct action at the polysomal level.

Specific inhibition of peptide-chain initiation by 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide.

M DMP (2-(4-methyl-2,6-dinitroanilino)-N-methyl- propionamide) is a potent inhibitor of mRNA-dependent amino acid incorporation in the in citro wheat embryo amino acid incorporating system. At concentrations above 5 X lo-,) hi, TMV-RNA-directed peptide synthesis is inhibited more than 957& Amino acid incorporation by polyribo- bomes (obtained from germinating wheat embryos) is not affected by MDMP. Similarly, polyuridylic acid directed polyphenylalanine synthesis is not affected by MDMP. These data indicate that MDMP inhibits peptide-chain initiation and does not affect peptide chain elongation. More precise localization of the MDMP inhibition is obtained by studying the effect of the inhibitor on the initiation reactions involved M uch progress has been made in elucidating the scheme of protein chain initiation in eukaryotic organisms. The over- all sequence of mRNA, initiator Met-tRNA and ribosomal subunit involvement has been clearly defined (Crystal et al., 1971; Prichard et al., 1971; Heywood and Thompson, 1971; Pragnell et al., 1971 ; Hoerz and McCarty, 1971 ; Burgess and Mach, 1971 ; Weeks et al., 1972) while the isolation and char- acterization of specific factors required for the constitutive reactions are well advanced (Crystal et a/., 1971 ; Heywood and Thompson, 1971 ; Seal et a/., 1972). Detailed anaylses of the individual partial reactions of initiation can be greatly facilitated by the availability of inhibitors with strict speci- ficity, such as those which have been used to great advantage in delineating the partial reactions of procaryotic protein synthesis (Pestka, 1971). Presently, however, the number of inhibitors available which specifically interfere with known steps of eukaryotic peptide-chain initiation is quite limited (i.e., aurintricarboxylic acid (Stewart et al., 1971 ; Marcus et a/., 1970a), edeine (Obrig et al., 1971), NaF (Hoerz and McCarty, 1971), and pactamycin (MacDonald and Gold- berg, 1970; Seal and Marcus, 1972)l. We now report a new inhibitor of eukaryotic peptide-chain initiation, 2-(4-methyl-

The activity of ribosomes whose RNA has been degraded by incubation in the presence or absence of oxidized glutathione

1. All of the RNA in ribosomes which have been incubated anaerobically for 24 h at 37° has been degraded to fragments which sediment as two broad peaks to the 14-S and 5-S regions of sucrose density gradients. These ribosomes are fully active when assayed for polyuridylic acid-directed polyphenylalanine synthesis. 2. Ribosomes incubated as above, but in the presence of 0.05 M oxidized glutathione, are no longer active in polypeptide synthesis and are completely dissociated into 30-S and 50-S subunits. The 30-S subunits are able to bind polyuridylic acid but cannot bind phenylalanyl-tRNA. The RNA of these ribosomes is also degraded to fragments of various sizes, none of which sediment slower than about 5 S.

Hybrid ribosome formation from Escherichia coli and chloroplast ribosome subunits.

A 70S ribosome was prepared from a 30S ribosome subunit from Euglena gracilis chloroplasts and a 50S ribosome subunit from Escherichia coli. This hybrid ribosome was active in polyuridylic acid-directed polyphenylalanine synthesis.

Defective ribosomes in chloramphenicol-treated Escherichia coli

Escherichia coli Q13 cells were treated with chloramphenicol for varying periods of time. Upon removal of this drug, the cells exhibited a lag period before the resumption of the normal rate of protein synthesis. This lag period was approximately proportional to the time the cells had been exposed to the drug. Treatment with chloramphenicol not only inhibited protein synthesis but also impaired the protein-synthesizing system of the cells. Ribosomes isolated from the cells exposed to the drug for more than 90 minutes lost 12 to 14% of their protein. When the ribosomes were washed with 1 m-NH 4Cl, no difference in protein content between the ribosomes from normal and drug-treated cells was detected. The ribosomes from drug-treated cells showed a marked decrease in bacteriophage MS2 RNA-specific binding of N-formyknethionyl-tRNA and MS2 RNA-directed protein synthesis when compared to normal ribosomes. No decrease in polyuridylic acid-directed polyphenylalanine synthesis was observed. Supernatant protein fraction from drug-treated cells did not appear to be defective. Addition of crude initiation protein factors prepared from normal ribosomes restored the ability of ribosomes from drug-treated cells to initiate and translate MS2 RNA normally. Acrylamide gel electrophoretic analysis indicated that initiation factor F2 was substantially decreased in the ribosomes from ehloramphenicol-treated cells. The protein fractions removed from ribosomes of normal and drug-treated cells by washing with 1 m-NH 4Cl were applied to DEAE-cellulose columns. Initiation factors F2 and F3 were either defective or substantially reduced in ribosomes from drug-treated cells. Addition of F2 and F3 derived from normal ribosomes restored the ability of the ribosomes from drug-treated cells to carry out MS2 RNA-directed protein synthesis.

Polypeptide synthesis with ribonuclease-digested ribosomes

An insoluble ribonuclease derivative has been used to digest Escherichia coli 50-S ribosomal subunits. The first scission of the 23-S ribosomal RNA appears to be specific and yields two unequal-sized fragments. Digestion by ribonuclease results in a loss of activity of the subunit in polyuridylic acid-directed polyphenylalanine synthesis. However, by carrying out the incubation with a higher concentration of divalent cations, polyphenylalanine synthesis could be demonstrated using ribosomes which did not contain any intact 23-S ribosomal RNA.

Mode of action of siomycin.

The incorporation of H3-lysine into Bacillus subtilis cells ceased almost immediately after the addition of while the continued incorporation of C14-uridine and of C14-thymidine into the trichloroacetic acid-insoluble fraction of the cells was observed for at least a further 20 minutes. In bacterialcell free systems (Bacillus subtilis and Escherichia coli) siomycin inhibits polyadenylic acid directed polylysine synthesis and polyuridylic acid-directed polyphenylalanine synthesis. However, aminoacyl-transfer RNA synthesis was hardly affected. The soluble siomycin, monothiomalic acid-siomycin A, was found to have an inhibitory action on bacterial protein biosynthesis, similar to and to roughly the same extent as siomycin A. In contrast to bacterial protein synthesis, C14-leucine incorporation into rabbit reticulocyte hemoglobin was not significantly decreased by the addition of siomycin. Protein biosynthesis in a cell-free system from rabbit reticuloeytes was also found to be highly resistant to this antibiotic. These results are in harmony with the previous observation that siomycin showed little toxicity to mammals.

Stimulation and inhibition of polypeptide synthesis by streptomycin in ribosomal systems of Escherichia coli, programmed with various messengers

1. The action in vitro of streptomycin on polypeptide synthesis has been studied with cell-free extracts from streptomycin-sensitive and resistant Escherichia coli. Various messengers, such as turnip yellow mosaic virus RNA, alfalfa mosaic virus RNA, polyuridylic acid and polyadenylic acid were employed, and ribosomes still associated with their endogenous messenger(s) were also included. 2. Varying effects of streptomycin on ribosomes from streptomycin-sensitive cells with messengers of viral origin and endogenous messenger(s) have been recorded. Depending on the Mg 2+ concentration either stimulation or inhibition was found. 3. Polyuridylic acid-directed polyphenylalanine synthesis and polyadenylic acid-induced polylysine formation were generally inhibited by streptomycin at all Mg 2+ concentrations studied. 4. The effects exerted by streptomycin on ribosomal systems from streptomycin-resistant cells were usually small or negligible. 5. Stimulation of “sensitive” systems was exerted on a ribosomal site. 6. Streptomycin-action became apparent during protein synthesis immediately after the antibiotic was added. The degree of inhibition or stimulation was the same whether the antibiotic was added before or during the process of protein synthesis. 7. The results are discussed in relation to current concepts of streptomycin action.