A direct evidence for the involvement of poly(A) in protein synthesis

A radioactive polyadenylated globin mRNA was translated in either rabbit reticulocyte lysate or wheat germ extract under various conditions. When globin mRNA was translated, globin synthesis was directly proportional to the rate of loss in A units from the poly(A) tail. On the other hand, when globin poly(A) mRNA was incubated under non-translated conditions, no loss of A units was detected. The presence of ribonuclease inhibitor in the reaction mixture did not alter either the rate of globin synthesis or the loss in A units from the poly(A) tail. The present data suggests a correlation between protein synthesis and loss in A units from the poly(A) tail.     

Synthesis of poly(A)-containing RNA in outgrowing spores of Bacillus subtilis

The synthesis of poly(A)-containing RNA in outgrowing spores of Bacillus subtilis was studied. A significant amount of RNA puls-labelled with 3H-uridine is polyadenylated. With the beginning of RNA synthesis in outgrowing spores labelled poly(A)-containing RNA was detected. The amount of poly(A)-RNA during the outgrowth and first cell division remains constant. Besides poly(A)-RNA the synthesis of tRNA and rRNA occurs. These results indicate a simultaneous activation of synthesis of tRNA, rRNA as well as of poly(A)-containing RNA during outgrowth of B. subtilis spores.     

Poliovirus Infection and Poly(A) Sequences of Cytoplasmic Cellular RNA

The influence of polio infection on Poly(A) sequences of cellular cytoplasmic RNA was investigated. In the presence of guanidine, cellular protein synthesis was still shut off after poliovirus infection, although there was no viral RNA synthesis. The Poly(A) of these cells was unchanged with respect to quantity, size, and linkage to cellular cytoplasmic RNA. This finding strongly suggests that the shut off of cellular protein synthesis is not caused by a change of the Poly(A) sequences of cellular mRNA.     

Adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate are synthesized by yeast acetyl coenzyme A synthetase.

Yeast (Saccharomyces cerevisiae) acetyl coenzyme A (CoA) synthetase (EC 6.2.1.1) catalyzes the synthesis of adenosine 5'-tetraphosphate (P4A) and adenosine 5'-pentaphosphate (p5A) from ATP and tri- or tetrapolyphosphate (P3 or P4), with relative velocities of 7:1, respectively. Of 12 nucleotides tested as potential donors of nucleotidyl moiety, only ATP, adenosine-5'-O-[3-thiotriphosphate], and acetyl-AMP were substrates, with relative velocities of 100, 62, and 80, respectively. The Km values for ATP, P3, and acetyl-AMP were 0.16, 4.7, and 1.8 mM, respectively. The synthesis of p4A could proceed in the absence of exogenous acetate but was stimulated twofold by acetate, with an apparent Km value of 0.065 mM. CoA did not participate in the synthesis of p4A (p5A) and inhibited the reaction (50% inhibitory concentration of 0.015 mM). At pH 6.3, which was optimum for formation of p4A (p5A), the rate of acetyl-CoA synthesis (1.84 mumol mg-1 min-1) was 245 times faster than the rate of synthesis of p4A measured in the presence of acetate. The known formation of p4A (p5A) in yeast sporulation and the role of acetate may therefore be related to acetyl-CoA synthetase.     

Application of lipase-catalyzed transformations for the synthesis of insect pheromones and related compounds

This review describes efficient means of preparing optically pure insect pheromones and related compounds via lipase-catalyzed enantioselective reaction on a large scale. (1) A new synthesis of the Japanese beetle pheromone, (R,Z)-(−)-5-(1-decenyl)oxacyclopentan-2-one, established by a combination of two lipase-catalyzed transformation was demonstrated. (2) A chemico-enzymatic procedure for the syntheses of both enantiomers of cupreous chafer beetle pheromone, (R,Z)- and (S,Z)-5-(1-octenyl)oxacyclopentan-2-one, was described. (3) An optical resolution of (±)-2,3-epoxy-8-methyl-1-nonanol, the key intermediate of the synthesis of gypsy moth pheromone, was demonstrated. (4) A practical chemico-enzymatic synthesis of (+)-disparlure in large scale was demonstrated. (5) A facile synthesis of carboxyalkyl acrylate, which is special monomers in the synthesis of the new polymers, by two lipase-catalyzed regioselective reactions was described.     

A preferential role for lysyl-tRNA4 in the synthesis of diadenosine 5',5'-P1,P4-tetraphosphate by an arginyl-tRNA synthetase-lysyl-tRNA synthetase complex from rat liver

The synthesis of diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) can be catalyzed in vitro by a tetrameric tRNA synthetase complex from rat liver containing two lysyl-tRNA synthetase and two arginyl-tRNA synthetase subunits. This reaction required ATP, AMP, 50-100 microM zinc, and inorganic pyrophosphatase. We show here that AMP can be omitted from the reaction and that the zinc levels can be markedly reduced provided catalytic amounts of tRNA(Lys) are added to the reaction mixture. Ap4A synthesis with purified tRNA(Lys) isoacceptors showed that the minor species, tRNA(4Lys), was 3-fold more active than either of the two major tRNA(Lys) species, tRNA(2Lys) and tRNA(5Lys). No activity could be demonstrated with tRNA(Lys) from Escherichia coli or with tRNA(Lys) or tRNA(Phe) from yeast. Aminoacylation of tRNA(4Lys) was strictly required as determined by the fact that Ap4A synthesis was not observed until aminoacylation was nearly complete, inhibitors of aminoacylation blocked Ap4A synthesis, and there was a strict requirement for added lysine. None of the above observations could be demonstrated, however, when lysyl-tRNA(Lys) was directly supplied to the reaction mixture. Optimum Ap4A synthesis was obtained by the addition of 1 mol of tRNA(Lys)/mol of the synthetase complex. This reaction is unique because it does not require the prior formation of an aminoacyl-AMP intermediate and because it can actively synthesize Ap4A at physiological zinc concentrations. The preferential role for tRNA(4Lys) in Ap4A synthesis is consistent with its prior implication in cell division.     

Chloroplast ribosomal ribonucleic acid synthesis in cultured spinach leaf tissue

Chloroplast rRNA synthesis was studied in spinach leaf tissue cultured under sterile conditions which eliminate bacterial rRNA synthesis. The synthesis was inhibited by darkness, but concomitant cytoplasmic rRNA synthesis was unaffected. A complex pattern of labelled rRNA precursors was found in extracts from cultured leaf tissue by using polyacrylamide-gel electrophoresis. However, differences between the precursor profiles of leaf tissue cultured in the light and in the dark could not be correlated with chloroplast rRNA synthesis since large amounts of high-molecular-weight precursors of cytoplasmic rRNA dominated the pattern in both cases. A double-isotope-labelling technique was used, which enabled light-stimulated rRNA synthesis to be studied in whole leaf tissue. Two rapidly labelled RNA species of molecular weights 1.15x10(6) and 0.65x10(6) were detected, which were thought to have possible precursor significance in the synthesis of mature chloroplast rRNA of molecular weights 1.04x10(6) and 0.56x10(6) respectively. Cycloheximide treatment resulted in the accumulation of RNA of molecular weight 1.8x10(6), whose function is unknown.     

Inhibition of host protein synthesis by vaccinia virus: fate of cell mRNA and synthesis of small poly (A)-rich polyribonucleotides in the presence of actinomycin D.

Purified vaccinia virus rapidly inhibited HeLa cell protein synthesis in the presence of actinomycin D. Under these conditions host polyribosomes were extensively degraded but the mRNA was stable as indicated by a greater than 90% recovery of prelabeled polyadenylylated RNA. Although actinomycin D prevented the synthesis of host mRNA and poly(A) in uninfected cells, incorporation of adenosine into poly(A) was inhibited by less than 50% in infected cells. Further analysis indicated that there was little or no normal size viral mRNA but that a unique class of small poly(A)-rich RNA was made in the presence of actinomycin D. From measurements of the RNase resistance and base composition of the RNA, approximately 40% of the nucleotide sequence was estimated to be poly(A). The poly(A)-rich RNA was found associated with small polyribosomes and monoribosomes that were inactive in protein synthesis. It was suggested that the poly(A) segment of the RNA is formed by the poly(A) polymerase previously found in vaccinia virus cores and that the inactive RNA, by competing with host mRNA, may contribute to the virus-mediated inhibition of host protein synthesis observed in the presence of actinomycin D.     

Synthesis of A7,B7-dicarbainsulin, an analogue with a noncleavable bond between A- and B-chain. II. Synthesis of the A-chain segments

As part of the total synthesis of [A7,B7-L,L-2,7-diaminosuberoyl]-des-(B26-B30)-insulin B25-amide, an insulin analogue containing a non-cleavable bond between A- and B-chain, the chemical synthesis of the A-chain segments is described. The N-terminal sequence A(1-6), Boc-Gly-Ile-Val-Glu(OBut)-Gln-Cys(SBut)-NH-NH2, was synthesized in solution. The middle segment A(8-16), Ddz-Thr(But)-Ser(But)-Ile-Cys(SBut)-Ser(But)-Leu-Tyr- (But)-Gln-Leu-NH-NH2, was obtained by solid phase synthesis according to the Fmoc strategy. The C-terminal segment A(17-21), Bpoc-Glu(OBut)-Asn-Tyr-Cys(Acm)-Asn-OBut, was prepared in solution.     

Chemotactic responses of human polymorphonuclear leukocytes to cyclic GMP and other compounds

Some metabolic properties of small molecular weight nuclear RNA (snRNA) components have been studied in human lymphocytes cultured with PHA. Pulse-labelling experiments with ³H-uridine in 3 h-intervals around the onset of DNA synthesis showed no qualitative or quantitative differences in the snRNA labelling pattern. Long labelling experiment with ³H-methionine demonstrated the following relative degrees of methylation: tRNA (1.0), 5S RNA (0), D (0.3), 5.5S RNA (0.2), C (0.6), A (0.2), L (0) and rRNA (0.2). Chase-experiments with ³H-methionine showed that the snRNA components D, C and A are metabolically stable with half-lives of not less than 30 h. Actinomycin D (0.05 μg/ml) reduced markedly the synthesis of rRNA and 5 S RNA whereas the synthesis of D, C, A and L was unaffected or only slightly affected. Actinomycin D at a concentration of 0.25 μg/ml inhibited the synthesis of D, C and A. Cycloheximide (0.19 μg/ml) reduced the synthesis of D, C and rRNA to about 50% of control whereas 5S RNA synthesis was only slightly inhibited and tRNA synthesis was unaffected.     

4-Coumarate:coenzyme A ligase has the catalytic capacity to synthesize and reuse various (di)adenosine polyphosphates

4-Coumarate:coenzyme A ligase (4CL) is known to activate cinnamic acid derivatives to their corresponding coenzyme A esters. As a new type of 4CL-catalyzed reaction, we observed the synthesis of various mono- and diadenosine polyphosphates. Both the native 4CL2 isoform from Arabidopsis (At4CL2 wild type) and the At4CL2 gain of function mutant M293P/K320L, which exhibits the capacity to use a broader range of phenolic substrates, catalyzed the synthesis of adenosine 5'-tetraphosphate (p(4)A) and adenosine 5'-pentaphosphate when incubated with MgATP(-2) and tripolyphosphate or tetrapolyphosphate (P(4)), respectively. Diadenosine 5',5',-P(1),P(4)-tetraphosphate represented the main product when the enzymes were supplied with only MgATP(2-). The At4CL2 mutant M293P/K320L was studied in more detail and was also found to catalyze the synthesis of additional dinucleoside polyphosphates such as diadenosine 5',5'-P(1),P(5)-pentaphosphate and dAp(4)dA from the appropriate substrates, p(4)A and dATP, respectively. Formation of Ap(3)A from ATP and ADP was not observed with either At4CL2 variant. In all cases analyzed, (di)adenosine polyphosphate synthesis was either strictly dependent on or strongly stimulated by the presence of a cognate cinnamic acid derivative. The At4CL2 mutant enzyme K540L carrying a point mutation in the catalytic center that is critical for adenylate intermediate formation was inactive in both p(4)A and diadenosine 5',5',-P(1),P(4)-tetraphosphate synthesis. These results indicate that the cinnamoyl-adenylate intermediate synthesized by At4CL2 not only functions as an intermediate in coenzyme A ester formation but can also act as a cocatalytic AMP-donor in (di)adenosine polyphosphate synthesis.     

Differential effects of concanavalin A, serum and cytokines on proteoglycan elaboration and DNA synthesis by mononuclear cells from human peripheral blood.

Human blood derived mononuclear cell (MC) cultures required concanavalin A (Con A) stimulation to synthesize and secrete into the medium high levels of a protease-resistant proteoglycan (PG) containing predominantly chondroitin sulfate (CS), which was elaborated largely by T-cells in culture. PG and DNA synthesis were studied in MC cultures in the absence and presence of Con A as well as serum and some biologically active polypeptide factors. In the presence of Con A, stimulation of PG synthesis was substantially greater in T-cell enriched cultures than in B-cell enriched cultures. DNA synthesis was also stimulated in the presence of Con A. This stimulation was concentration-dependent, but required the presence of serum for additional responses. DNA and cell proliferation were stimulated by interleukin-2 (IL-2), but PG production was not stimulated by conditioned media, IL-1, IL-2, IL-3, or transforming growth factor-beta (TGF-beta). Our results indicate that the elaboration of PG from T-cells of human MC is independent of the effects of regulatory peptides on cell proliferation and DNA synthesis.     

Regulation by interleukin 2 of interleukin 2 receptors and gamma-interferon synthesis by human thymocytes: augmentation of interleukin 2 receptors by interleukin 2

The role of interleukin 2 (IL 2) on the expression of IL 2 receptors and on the synthesis of gamma-interferon (gamma-IFN) by human thymocytes was investigated. Human thymocytes isolated from specimens obtained during cardiac surgery of infants and children were induced with one or all of the following agents: IL 2, concanavalin A (Con A), and 12-O-tetradecanoylphorbol 13-acetate (TPA). The expression of IL 2 receptors and gamma-IFN titers were determined. The results indicate that thymocytes cultured in complete medium do not express receptors for IL 2, nor did IL 2 by itself induce the expression of IL 2 receptors. Con A induced the expression of IL 2 receptors by a moderate number of the thymocyte population and induced the synthesis of low amounts of gamma-IFN. Preincubation of thymocytes with TPA increased the response to Con A; both the number of thymocytes expressing receptors and the synthesis of gamma-IFN were increased. Addition of IL 2 to these cultures further augmented the expression of IL 2 receptors and gamma-IFN synthesis and resulted in the optimal expression of IL 2 receptors and maximal gamma-IFN synthesis. The expression of IL 2 receptors could be detected within 24 hr and preceded the induction of proliferation; it was therefore probably not due to the clonal expansion of a population of receptor-bearing thymocytes. Conversely, inhibition of IL 2 synthesis with dexamethasone (Dex) by thymocytes activated with Con A, or inhibition of the function of IL 2 receptors by anti-Tac, resulted in a decrease in the number of IL 2 receptor-bearing thymocytes activated with Con A, or inhibition of the function of IL 2 receptors by anti-Tac, resulted in a decrease in the number of IL 2 receptor-bearing thymocytes and of gamma-IFN synthesis. Thymocytes activated with TPA and Con A were more resistant to the inhibitory effects of Dex on the expression of IL 2 receptors than thymocytes activated with Con A alone. Maximal inhibition of the expression of IL 2 receptors and of gamma-IFN synthesis was achieved as a result of the synergistic effect of anti-Tac with Dex. Therefore, when IL 2 was prevented from binding to the receptors, and IL 2 synthesis was inhibited, the number of thymocytes expressing IL 2 receptors was sharply reduced and gamma-IFN synthesis was markedly inhibited.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reversal of the deoxyhypusine synthesis reaction. Generation of spermidine or homospermidine from deoxyhypusine by deoxyhypusine synthase

Deoxyhypusine synthase catalyzes the first step in hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine) synthesis in a single cellular protein, eIF5A precursor. The synthesis of deoxyhypusine catalyzed by this enzyme involves transfer of the 4-aminobutyl moiety of spermidine to a specific lysine residue in the eIF5A precursor protein to form a deoxyhypusine-containing eIF5A intermediate, eIF5A(Dhp). We recently discovered the efficient reversal of deoxyhypusine synthesis. When eIF5A([3H]Dhp), radiolabeled in the 4-aminobutyl portion of its deoxyhypusine residue, was incubated with human deoxyhypusine synthase, NAD, and 1,3-diaminopropane, [3H]spermidine was formed by a rapid transfer of the radiolabeled 4-aminobutyl side chain of the [3H]deoxyhypusine residue to 1,3-diaminopropane. No reversal was observed with [3H]hypusine protein, suggesting that hydroxylation at the 4-aminobutyl side chain of the deoxyhypusine residue prevents deoxyhypusine synthase-mediated reversal of the modification. Purified human deoxyhypusine synthase also exhibited homospermidine synthesis activity when incubated with spermidine, NAD, and putrescine. Thus it was found that [14C]putrescine can replace eIF5A precursor protein as an acceptor of the 4-aminobutyl moiety of spermidine to form radiolabeled homospermidine. The Km value for putrescine (1.12 mM) as a 4-aminobutyl acceptor, however, is much higher than that for eIF5A precursor (1.5 microM). Using [14C]putrescine as an acceptor, various spermidine analogs were evaluated as donor substrates for human deoxyhypusine synthase. Comparison of spermidine analogs as inhibitors of deoxyhypusine synthesis, as donor substrates for synthesis of deoxyhypusine (or its analog), and for synthesis of homospermidine (or its analog) provides new insights into the intricate specificity of this enzyme and versatility of the deoxyhypusine synthase reaction.     

Poly(A)+ RNA metabolism during oogenesis in Xenopus laevis.

In this study, we have measured the synthesis and turnover of oligo(dT)cellulose-bound RNA [poly(A)⁺ RNA] in Xenopus laevis oocytes at the maximal lampbrush chromosome stage (stage 3) and at the completion of oocyte growth (stage 6). Oocytes at both stages are shown to be active in the synthesis of poly(A)⁺ RNA. In stage 6 oocytes, the mean rate of synthesis of stable poly(A)⁺ RNA is 15% the instantaneous rate of synthesis, while the mean half-life of the unstable component is 1.6 hr. In contrast, the instantaneous rate of synthesis in stage 3 oocytes is about one-third that seen in stage 6, and most of it is devoted to the production of unstable species with an average half-life of 5 hr. Studies on the nuclear versus the cytoplasmic distribution of the newly synthesized poly(A)⁺ RNA demonstrated that by the end of a 12-hr labeling period for stage 3 oocytes and a 24-hr labeling period for stage 6 oocytes, approximately half of the material was cytoplasmic. This cytoplasmic material had the same electrophoretic mobility as bulk poly(A)⁺ RNA. Similarly, as with bulk poly(A)⁺ RNA, little, if any, of the newly synthesized material was found to be polysomal. Also, poly(A) labeling studies indicated that the newly synthesized poly(A)⁺ RNA was associated with the synthesis of poly(A) of the same length as that appearing on bulk poly(A)⁺ RNA. Studies on the content of bulk oligo(dT)cellulose-bound RNA indicated that about 86 ng is present in both stage 3 and stage 6 oocytes. The continual synthesis of poly(A)⁺ RNA throughout oogenesis in the absence of its accumulation led to the conclusion that it must be turning over. These data are discussed in relation to the hypothesis that bulk levels of poly(A)⁺ RNA are maintained by continually changing rates of synthesis and degradation.     

Inhibition of Interjacent Ribonucleic Acid (26S) Synthesis in Cells Infected by Sindbis Virus

The interrelationship of viral ribonucleic acid (RNA) and protein synthesis in cells infected by Sindbis virus was investigated. When cultures were treated with puromycin early in the course of infection, the synthesis of interjacent RNA (26S) was preferentially inhibited. A similar result was obtained by shifting cells infected by one temperature-sensitive mutant defective in RNA synthesis from the permissive (29 C) to the nonpermissive (41.5 C) temperature. Under both conditions, the viral RNA produced appeared to be fully active biologically. Once underway, the synthesis of viral RNA in wild-type Sindbis infections did not require concomitant protein synthesis.     

A1Ao-ATP synthase of Methanobrevibacter ruminantium couples sodium ions for ATP synthesis under physiological conditions

An unresolved question in the bioenergetics of methanogenic archaea is how the generation of proton-motive and sodium-motive forces during methane production is used to synthesize ATP by the membrane-bound A(1)A(o)-ATP synthase, with both proton- and sodium-coupled enzymes being reported in methanogens. To address this question, we investigated the biochemical characteristics of the A(1)A(o)-ATP synthase (MbbrA(1)A(o)) of Methanobrevibacter ruminantium M1, a predominant methanogen in the rumen. Growth of M. ruminantium M1 was inhibited by protonophores and sodium ionophores, demonstrating that both ion gradients were essential for growth. To study the role of these ions in ATP synthesis, the ahaHIKECFABD operon encoding the MbbrA(1)A(o) was expressed in Escherichia coli strain DK8 (Δatp) and purified yielding a 9-subunit protein with an SDS-stable c oligomer. Analysis of the c subunit amino acid sequence revealed that it consisted of four transmembrane helices, and each hairpin displayed a complete Na(+)-binding signature made up of identical amino acid residues. The purified MbbrA(1)A(o) was stimulated by sodium ions, and Na(+) provided pH-dependent protection against inhibition by dicyclohexylcarbodiimide but not tributyltin chloride. ATP synthesis in inverted membrane vesicles lacking sodium ions was driven by a membrane potential that was sensitive to cyanide m-chlorophenylhydrazone but not to monensin. ATP synthesis could not be driven by a chemical gradient of sodium ions unless a membrane potential was imposed. ATP synthesis under these conditions was sensitive to monensin but not cyanide m-chlorophenylhydrazone. These data suggest that the M. ruminantium M1 A(1)A(o)-ATP synthase exhibits all the properties of a sodium-coupled enzyme, but it is also able to use protons to drive ATP synthesis under conditions that favor proton coupling, such as low pH and low levels of sodium ions.     

Long functionalized poly(ethylene glycol)s of defined molecular weight: synthesis and application in solid-phase synthesis of conjugates

A concise synthesis of long-chain poly(ethylene glycol) (PEG) of defined molecular weight up to 29 ethyleneoxy units is described. These PEG diols were converted in a two-step synthesis into Fmoc-protected PEG amino acids, suitable as long linkers and compatible with solid-phase peptide synthesis. Long PEG chains (MW > 1000) can be readily synthesized with this method, which has the advantage of defined single molecular weight products over the comparable commercial polymers. The application of these PEG linkers to the synthesis of peptide-PEG-folate conjugates on a solid support was investigated. A method for the solid support synthesis of the targeting component of the conjugate, folic acid-cysteine, was developed, resulting in improved yields with respect to literature methods. The assembly of the peptide, PEG linker, and targeting group on solid support resulted in the synthesis of a conjugate of defined molecular weight and structure.     

Effect of hairpin DNA on poly(phenylalanine) synthesis.

In a poly(U) dependent poly(Phe) synthesis, an unaminoacylated tRNA(phe) binds to a ribosome and inhibits poly(Phe) synthesis in excess tRNA(Phe). A small DNA fragment having a hairpin structure was added to this system and was found to prevent an unaminoacylated tRNA(phe) from binding to a ribosome and enhance the efficiency of poly(Phe) synthesis in the presence of excess tRNA(phe).