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Remdesivir is an antiviral drug initially designed against the Ebola virus. The results obtained with it both in biochemical studies in vitro and in cell line assays in vivo were very promising, but it proved to be ineffective in clinical trials. Remdesivir exhibited far better efficacy when repurposed against SARS-CoV-2. The chemistry that accounts for this difference is the subject of this study. Here, we examine the hypothesis that remdesivir monophosphate (RMP)-containing RNA functions as a template at the polymerase site for the second run of RNA synthesis, and as mRNA at the decoding center for protein synthesis. Our hypothesis is supported by the observation that RMP can be incorporated into RNA by the RNA-dependent RNA polymerases (RdRps) of both viruses, although some of the incorporated RMPs are subsequently removed by exoribonucleases. Furthermore, our hypothesis is consistent with the fact that RdRp of SARS-CoV-2 selects RMP for incorporation over AMP by 3-fold in vitro , and that RMP-added RNA can be rapidly extended, even though primer extension is often paused when the added RMP is translocated at the i + 3 position (with i the nascent base pair at an initial insertion site of RMP) or when the concentrations of the subsequent nucleoside triphosphates (NTPs) are below their physiological concentrations. These observations have led to the hypothesis that remdesivir might be a delayed chain terminator. However, that hypothesis is challenged under physiological concentrations of NTPs by the observation that approximately three-quarters of RNA products efficiently overrun the pause.

Nonsense mutations that result in premature stop codons in the HBB gene cause β-thalassemia. This disease is characterized by a reduced hemoglobin level due to the lack of β-globin. Compounds that induce translational readthrough across the thalassemia-causing premature stop codon will have therapeutic benefits. Currently available molecules that induce translational readthrough lack specificity, and some of them show toxicity after prolonged use. In this study, we have developed an oligonucleotide-based approach to induce translational readthrough across the thalassemia-causing premature stop codon. Oligonucleotides that target HBB mRNA downstream of the premature stop codon could induce translational readthrough, generating a full-length β-globin protein. We show this effect using fluorescence- and luminescence-based readthrough assays and by Western blot. Remarkably, the amount of oligonucleotide-induced translational readthrough product is comparable to that of the protein generated by normal translation when there was no premature stop codon. Thus, these oligonucleotides, with certain modifications, have the potential to be used as drugs for the treatment of β-thalassemia. Also, this strategy can be extended to treat other genetic diseases caused by premature stop codons.

The threat of bacteria resistant to multiple antibiotics poses a major public health problem requiring immediate and coordinated action worldwide. While infectious pathogens have become increasingly resistant to commercially available drugs, antibiotic discovery programs in major pharmaceutical companies have produced no new antibiotic scaffolds in 40 years. As a result, new strategies must be sought to obtain a steady supply of novel scaffolds capable of countering the spread of resistance. The bacterial ribosome is a major target for antimicrobials and is inhibited by more than half of the antibiotics used today. Recent studies showing that the ribosome is a target for several classes of ribosomally synthesized antimicrobial peptides point to ribosome-targeting peptides as a promising source of antibiotic scaffolds. In this Perspective, we revisit the current paradigm of antibiotic discovery by proposing that the bacterial ribosome can be used both as a target and as a tool for the production and selection of peptide-based antimicrobials. Turning the ribosome into a high-throughput platform for the directed evolution of peptide-based antibiotics could be achieved in different ways. One possibility would be to use a combination of state-of-the-art microfluidics and genetic reprogramming techniques, which we will review briefly. If it is successful, this strategy has the potential to produce new classes of antibiotics for treating multi-drug-resistant pathogens.

We have examined the abilities of three complementary γ-peptide nucleic acid (γPNA) oligomers to invade an RNA G-quadruplex and potently inhibit translation of a luciferase reporter transcript containing the quadruplex-forming sequence (QFS) within its 5'-untranslated region. All three γPNA oligomers bind with low nanomolar affinities to an RNA oligonucleotide containing the QFS. However, while all probes inhibit translation with low to midnanomolar IC50 values, the γPNA designed to hybridize to the first two G-tracts of the QFS and adjacent 5'-overhanging nucleotides was 5-6 times more potent than probes directed to either the 3'-end or internal regions of the target at 37 °C. This position-dependent effect was eliminated after the probes and target were preincubated at an elevated temperature prior to translation, demonstrating that kinetic effects exert significant control over quadruplex invasion and translation inhibition. We also found that antisense γPNAs exhibited similarly potent effects against luciferase reporter transcripts bearing QFS motifs having G2, G3, or G4 tracts. Finally, our results indicate that γPNA oligomers exhibit selectivity and/or potency higher than those of other antisense molecules such as standard PNA and 2'-OMe RNA previously reported to target G-quadruplexes in RNA.

RNA G-quadruplex (GQ) structures act as regulators of a diverse array of cellular processes including translation, pre-mRNA processing, and mRNA targeting. We report here a strategy of harnessing the natural ability of RNA GQs to inhibit translation by rationally inducing a GQ on a targeted mRNA to knockdown endogenous gene expression. We chose to target eIF-4E because of its key role in translation initiation and overexpression in multiple cancers and with the expectation that downregulation of eIF-4E would result in antiproliferation of cancer cells. Targeted hybrid (RNA:DNA) GQ structures were induced at the 5'-untranslated region (UTR) and the protein coding region of the eIF-4E mRNA by rationally designed and partially modified extraneous DNA sequences and their effect on eIF-4E expression was determined. The formation of a stable induced G-quadruplex was established by biophysical and biochemical methods. Thermodynamic parameters calculated from CD melting indicate formation of a stable induced GQ at a physiologically relevant salt concentration. We established the specificity and efficacy of the induced GQ formation by monitoring the targeted repression of a reporter gene. Most importantly we have demonstrated that inducing GQ in the 5'-UTR and the protein coding region of eIF-4E mRNA in human cancer cells results in 30% and 60% inhibition of the endogenous protein expression, respectively. Treating with the GQ inducing oligonucleotide sequences resulted in a decrease in the viability of human cancer cells in a dose-dependent manner. The above concept opens up a new strategy for targeted modulation of endogenous gene expression.

Viomycin belongs to the tuberactinomycin family of antibiotics against tuberculosis. However, its inhibition mechanism remains elusive. Although it is clear that viomycin inhibits the ribosome intersubunit ratcheting, there are contradictory reports about whether the antibiotic viomycin stabilizes the tRNA hybrid or classical state. By using a single-molecule FRET method to directly observe the tRNA dynamics relative to ribosomal protein L27, we have found that viomycin trapped the hybrid state within certain ribosome subgroups but did not significantly suppress the tRNA dynamics. The persistent fluctuation of tRNA implied that tRNA motions were decoupled from the ribosome intersubunit ratcheting. Viomycin also promoted peptidyl-tRNA fluctuation in the posttranslocation complex, implying that, in addition to acylated P-site tRNA, the decoding center also played an important role of ribosome locking after translocation. Therefore, viomycin inhibits translocation by trapping the hybrid state in the pretranslocation complex and disturbing the stability of posttranslocation complex. Our results imply that ribosome translocation is possibly a synergistic process of multiple decoupled local dynamics.

Whereas the transcription factors GATA-1 and GATA-2 function both uniquely and redundantly to control blood cell development, the process termed hematopoiesis, mechanisms underlying their unique versus common functions are poorly understood. We used two independent assays to demonstrate that GATA-1 is considerably more stable than GATA-2 in multiple cellular contexts, even though both factors are subject to degradation via the ubiquitin-proteasome system. Studies with GATA factor mutants and novel chimeric GATA factors provided evidence that both GATA-1 and GATA-2 have highly unstable zinc finger core modules. The GATA-1 and GATA-2 N-termini both confer stabilization to their respective zinc finger core modules. In contrast, the GATA-1 and GATA-2 C-termini confer stabilization and destabilization, respectively. As GATA-2 stabilization via proteasome inhibition impairs the capacity of GATA-1 to displace GATA-2 from endogenous chromatin sites, we propose that differential GATA factor stability is an important determinant of chromatin target site occupancy and therefore the establishment of genetic networks that control hematopoiesis.

Death-associated protein kinase (DAPK) is a pro-apoptotic, calcium/calmodulin-regulated protein kinase that is a drug discovery target for neurodegenerative disorders. Despite the potential profound physiological role of DAPK in neuronal function and pathophysiology, the endogenous substrate(s) of this kinase and the mechanisms via which DAPK elicits its biological action remain largely unknown. We report here that the mammalian 40S ribosomal protein S6 is a DAPK substrate. Results from immunoprecipitation experiments are consistent with endogenous DAPK being associated with endogenous S6 in rat brain. When S6 is a component of the 40S ribosomal subunit complex, DAPK selectively phosphorylates it at serine 235, one of the five sites in S6 that are phosphorylated by the S6 kinase family of proteins. The amino acid sequence flanking serine 235 matches the established pattern for DAPK peptide and protein substrates. Kinetic analyses using purified 40S subunits revealed a K(m) value of 9 microM, consistent with S6 being a potential physiological substrate of DAPK. This enzyme-substrate relationship has functional significance. DAPK suppresses translation in rabbit reticulocyte lysate, and treatment of neuroblastoma cells with a stimulator of DAPK reduces protein synthesis. In both cases, suppression of translation correlates with increased phosphorylation of S6 at serine 235. These results demonstrate that DAPK is a S6 kinase and provide evidence for a novel role of DAPK in the regulation of translation.

Insulin has been shown to acutely regulate hepatic apolipoprotein B (apoB) secretion at both translational and post-translational levels; however, mechanisms of apoB mRNA translational control are largely unknown. Recent studies of apoB untranslated regions (UTRs) revealed a potentially important role for cis-trans interactions at the 5' and 3' UTRs. In the present paper, deletion constructs of the UTR regions of apoB revealed that the 5' UTR was necessary and sufficient for insulin to inhibit synthesis of apoB15. Metabolic radiolabeling and in vitro translation experiments in the presence of protease inhibitors confirmed that the effect of insulin on the apoB 5' UTR was translational in nature. Using the nondenaturing electrophoretic mobility shift assay (EMSA), protein-RNA complexes were detected binding to the apoB 5' and 3' UTRs. Denaturing EMSA identified a 110-kDa protein interacting at the 5' UTR. Nondenaturing EMSA determined that insulin altered binding of large protein complexes to the 5' UTR. Binding specificity was determined by competition with both specific and nonspecific competitors. Insulin treatment decreased binding of the 110-kDa protein to the 5' UTR as visualized by EMSA. Absence of insulin increased binding of this trans-acting factor to the 5' UTR by 2-fold. Analysis of the 3' UTR showed no significant insulin-mediated changes in binding of trans-acting factors. We thus propose the existence of a novel RNA-binding insulin-sensitive factor that binds to the 5' UTR and may regulate apoB mRNA translation. Perturbations in hepatic insulin signaling as observed in insulin-resistant states may alter cis-trans interactions at the 5' UTR, leading to alterations in the rate of apoB mRNA translation, thus contributing to apoB-lipoprotein overproduction.

The clinical use of tetracycline antibiotics has decreased due to the emergence of efflux and ribosomal protection-based resistance mechanisms. Currently in phase III clinical trials, the glycylcycline derivative tigecycline (GAR-936) containing a 9-tert-butylglycylamido group is part of a new generation of tetracycline antibiotics developed during the 1990s. Tigecycline displays a broad spectrum of antibacterial activity and circumvents the efflux and ribosomal protection resistance mechanisms. The TetX protein is a flavin-dependent monooxygenase that modifies first and second generation tetracyclines and requires NADPH, Mg(2+), and O(2) for activity. We report that tigecycline is a substrate for TetX and that bacterial strains containing the tet(X) gene are resistant to tigecycline. The resistance is due to the modification of tigecycline by TetX to form 11a-hydroxytigecycline, which we have shown has a weakened ability to inhibit protein translation compared with tigecycline. We have explored the basis of this decreased ability to block translation and found that hydroxylation occurs in the region of the molecule important for coordinating magnesium. 11a-Hydroxytigecycline forms a weaker complex with magnesium than tigecycline; the crystal structure of tetracycline in complex with the ribosome has shown that magnesium coordination is critical for binding tetracycline. Although tet(X) has not been isolated from any clinically resistant strains, our report demonstrates the first enzymatic resistance mechanism to tigecycline and provides an alert for the surveillance of resistant strains that may contain tet(X).

Glucocorticoids up-regulate the glucocorticoid receptor (GR) in the human T-lymphoblast cell line CEM-C7. One mechanism for the up-regulation of the GR protein is the well-known up-regulation of GR transcripts. We have investigated the effect of other factors on the up-regulation. At least three promoters (1A, 1B, and 1C) exist, which give rise to GR transcripts with different exon 1 sequences. Transcripts with different exon 1 sequences have similar stabilities. Glucocorticoids have little, if any, effect on mRNA stability. In transfection experiments of the GR-deficient mouse fibroblast cell line E8.2, different exon 1 sequences furthermore caused no significant differences in the translational efficiencies of GR transcripts. However, the ratio between the concentrations of the glucocorticoid receptor B (GR-B) isoform and the glucocorticoid receptor A (GR-A) isoform was higher for transcripts containing the exon 1A3 sequence arising from promoter 1A than in transcripts containing exon 1 sequences from promoters 1B and 1C. Because the GR-B isoform is more active in transactivation then GR-A, this would tend to fine-tune glucocorticoid responsiveness of CEM-C7 cells, which express exon 1A3-containing transcripts. We also found that glucocorticoids do not decrease the stability of the GR protein in CEM-C7 cells. In contrast to other cell lines that downregulate GR expression in response to glucocorticoids, CEM-C7 lymphoblasts possess three mechanisms ensuring high glucocorticoid responsiveness: an up-regulation of GR mRNA by glucocorticoids, no destabilization of GR protein by glucocorticoids, and a high activity of promoter 1A with concomitant high expression of the GR-B isoform.

Aminoglycoside antibiotics bind to the A-site decoding region of bacterial rRNA causing mistranslation and/or premature message termination. Aminoglycoside binding to A-site RNA decoding region constructs is established here to be only weakly stereospecific. Mirror-image prokaryotic A-site decoding region constructs were prepared in the natural D-series and the enantiomeric L-series and tested for binding to a series of aminoglycosides. In general, aminoglycosides bind to the D-series decoding region constructs with 2-3-fold higher affinities than they bind to the enantiomeric L-series. Moreover, L-neamine, the enantiomer of naturally occurring D-neamine, was prepared and shown to bind approximately 2-fold more weakly than D-neamine to the natural series decoding region construct, a result consistent with weakly stereospecific binding. The binding of naturally occurring D-neamine and its synthetic L-enantiomer was further evaluated with respect to binding to prokaryotic and eukaryotic ribosomes. Here, weak stereospecifcity was again observed with L-neamine being the more potent binder by a factor of approximately 2. However, on a functional level, unnatural L-neamine proved to inhibit in vitro translation with significantly lower potency (approximately 5-fold) than D-neamine. In addition, both L- and D-neamine are bacteriocidal toward Gram-(-) bacteria. L-Neamine inhibits the growth of E. coli and P. aeruginosa with 8- and 3-fold higher MIC than D-neamine. Interestingly, L-neamine also inhibits the growth of aminoglycoside-resistant E. coli, which expresses a kinase able to phosphorylate and detoxify aminoglycosides of the D-series. These observations suggest that mirror-image aminoglycosides may avoid certain forms of enzyme-mediated resistance.

Peptide nucleic acids (PNAs) are synthetic polynucleobases that bind to DNA and RNA with high affinity and specificity and with poor membrane permeability. Although PNAs have an enormous potential as antisense agents, the success of antisense PNA applications will require efficient cellular uptake. In this study, a unique antisense 14-mer anti-inducible nitric oxide synthase (iNOS) was encapsulated into erythrocytes (RBC) by hypotonic dialysis. RBC loaded with PNA (10.5 +/- 3.5 micromol/mL RBC) were targeted specifically to murine macrophages, taking advantage of an in vitro opsonization induced by ZnCl(2) and bis-sulfosuccynimidil-suberate (BS(3)). This in vitro opsonization enhanced the phagocytosis of loaded RBC and the delivery of PNA into macrophages (0.72 pmol/10(6) macrophages). The efficacy of this delivery system is demonstrated by decreases in NO production and iNOS protein expression inside the macrophage. Therefore, we can suggest this novel approach for biomedical application.

Antisense oligonucleotides have the potential to make revolutionary contributions to basic science and medicine. Oligonucleotides can bind mRNA and inhibit translation. Because they can be rapidly synthesized to be complementary to any sequence, they offer ideal tools for exploiting the massive amount of genome information now available. However, until recently, this potential was largely theoretical, and antisense experiments often produced inconclusive or misleading outcomes. This review will discuss the chemical and biological properties of some of the different types of oligomers now available and describe the challenges confronting in vitro and in vivo use of oligonucleotides. Oligomers with improved chemical properties, combined with advances in cell biology and success in clinical trials, are affording powerful new options for basic research, biotechnology, and medicine.

The effect of two photoreactive analogues of spermine, N(1)-azidobenzamidino- (ABA-) spermine and N(1)-azidonitrobenzoyl- (ANB-) spermine, on ribosomal functions was studied in a cell-free system derived from Escherichia coli. In the dark, both analogues stimulated the binding of AcPhe-tRNA to poly(U)-programmed ribosomes, enhanced the stability of the ternary complex AcPhe-tRNA.poly(U).ribosome (complex C), and caused stimulatory and inhibitory effects on peptidyltransferase activity. ABA-spermine exhibited more pronounced effects than ANB-spermine. Each photoprobe was covalently attached after irradiation to both ribosomal subunits and also to free rRNA isolated from 70S ribosomes. Photolabeled complex C showed a reactivity toward puromycin, similar to that exhibited by complex C reacting reversibly with photoprobes free in solution. The distribution of the incorporated radioactivity among the ribosomal components was determined under two experimental conditions, one stimulating and the other inhibiting peptidyltransferase activity. Under both conditions, ABA-spermine was the strongest cross-linker. Upon stimulatory conditions, 14% of ABA-[(14)C]spermine cross-linked to complex C was bound to the protein fraction. The proteins primarily labeled were identified as S3, S4, L2, L3, L6, L15, L17, and L18. Upon inhibitory conditions, a higher percent of the incorporated radioactivity was found in ribosomal proteins, while the pattern of protein labeling was characterized by a remarkable decrease of cross-linked proteins L2, L3, L6, L15, L17. and L18 and by an increase of cross-linked proteins S9, S18, L1, L16, L22, L23, and L27. On the basis of these results and literature data, the involvement of spermine in the conformation and important functions of ribosomes is discussed.

The antineoplastic cyclic depsipeptide didemnin B (DB) inhibits protein synthesis in cells and in vitro. The stage at which DB inhibits protein synthesis in cells is not known, although dehydrodidemnin B arrests translation at the stage of polypeptide elongation. Inhibition of protein synthesis by DB in vitro also occurs at the elongation stage, and it was shown previously that DB prevents EF-2-dependent translocation in partial reaction models of protein synthesis. This inhibition of translocation displays an absolute requirement for EF-1alpha; however, the dependence upon EF-1alpha was previously unexplained. It is shown here that DB binds only weakly to EF-1alpha/GTP in solution, but binds to ribosome. EF-1alpha complexes with a dissociation constant K(d) = 4 microM. Thus, the inhibition of protein synthesis by DB appears to involve an interaction with both EF-1alpha and ribosomes in which all three components are required. Using diphtheria toxin-mediated ADP-ribosylation to assay for EF-2, it is demonstrated that DB blocks EF-2 binding to pre-translocative ribosome.EF-1alpha complexes, thus preventing ribosomal translocation. Based on this model for protein synthesis inhibition by DB, and the proposed mechanism of action of fusidic acid, evidence is presented in support of the Grasmuk model for EF-1alpha function in which this elongation factor does not fully depart the ribosome during polypeptide elongation.

Fifty-eight analogues of the 5'-terminal 7-methylguanosine-containing cap of eukaryotic messenger RNA were synthesized and tested for their ability to inhibit in vitro protein synthesis. A new algorithm was developed for extracting KI, the dissociation constant for the cap analogue.eIF4E complex, from protein synthesis data. The results indicated that addition of a methyl group to the N2 of guanine produced more inhibitory compounds, but addition of a second methyl group to N2 decreased the level of inhibition dramatically. Aryl substitution at N7 improved the efficacy of guanine nucleoside monophosphate analogues. Substitution of the aromatic ring at the para position with methyl or NO2 groups abolished this effect, but substitution with Cl or F enhanced it. By contrast, aryl substitution at N7 in nucleoside di- or triphosphate analogues produced only minor effects, both positive and negative. By far the strongest determinants of inhibitory activity for cap analogues were phosphate residues. The beneficial effect of more phosphate residues was related more to anionic charge than to the number of phosphate groups per se. The second nucleotide residue in analogues of the form m7GpppN affected inhibitory activity in the order G > C > U > A, but there was no effect of 2'-O-modification. Opening the first ribose ring of m7GpppG analogues dramatically decreased activity, but alterations at the 2'-position of this ribose had no effect. Non-nucleotide-based cap analogues containing benzimidazole derivatives were inhibitory, though less so than those containing 7-methylguanine.

Onconase is a cytotoxic ribonuclease with antitumor properties. A semisynthetic gene encoding the entire protein sequence was constructed by fusing oligonucleotides coding for the first 15 and the last 6 of the 104 amino acids to a genomic clone that encoded the remaining amino acid residues [Newton, D. L., et al. (1997) Protein Eng. 10, 463-470]. The resulting protein product expressed in Escherichia coli exhibited little enzymatic or cytotoxic activity due to the unprocessed N-terminal Met amino acid residue. In this study, we demonstrate that modification of the 5'-region of the gene to encode [Met-(-1)]Ser or [Met-(-1)]Tyr instead of the native pyroglutamate results in recombinant onconase derivatives with restored activities. [Met-(-1)]rOnc(E1S) was more active than [Met-(-1)]rOnc(E1Y) in all assays tested. Consistent with the action of native onconase, [Met-(-1)]rOnc(E1S) was a potent inhibitor of protein synthesis in the cell-free rabbit reticulocyte lysate assay, degrading tRNA at concentrations that correlated with inhibition of protein synthesis. An interesting difference between the recombinant onconase derivatives and the native protein was their susceptibility to inhibition by the major intracellular RNase inhibitor, PRI (onconase is refractory to PRI inhibition). [Met-(-1)]rOnc(E1S) and [Met-(-1)]rOnc(E1Y) inhibited protein synthesis in intact SF539 neuroblastoma cells with IC50's very similar to that of onconase (IC50 3.5, 10, and 10 microg/mL after 1 day and 0.16, 0.35, and 2.5 microg/mL after 5 days for onconase, [Met-(-1)]rOnc(E1S), and [Met-(-1)]rOnc(E1Y), respectively). Similar to that of onconase, cytotoxic activity of the recombinant derivatives was potentiated by monensin, NH4Cl, and retinoic acid. Brefeldin A completely blocked the enhancement of cytotoxicity caused by retinoic acid with all three proteins. Thus, drug-induced alterations of the intracellular trafficking of the recombinant derivatives also resembles that of onconase. Stability studies as assessed in serum-containing medium in the presence or absence of cells at 37 degreesC showed that the recombinant proteins were as stable to temperature and cell culture conditions as the native protein. Therefore, exchanging the Glu amino acid residue at the amino terminus of onconase with an amino acid residue containing a hydroxyl group produces recombinant proteins with ribonuclease and cytotoxic properties similar to native onconase.

The 4E binding proteins (4E-BP1 and 4E-BP2) inhibit translation by binding to the limiting, proto-oncogenic initiation factor eIF4E. 4E-BPs produced in Escherichia coli had little or no folded structure, measured by NMR and CD. However, these proteins inhibited translation in reticulocyte lysate. Furthermore, they bound to isolated mouse eIF4E, showing a few broader, dispersed new NMR signals but no general increase in chemical shift dispersion. A peptide with the sequence of 4E-BP1 residues 49-68 was sufficient to bind eIF4E and to inhibit translation in reticulocyte lysate. These results suggest that a short central region of the 4E-BPs is responsible for eIF4E binding and translation inhibition while the remainder is unfolded and flexible.

The proteasome inhibitors, lactacystin and N-acetyl-leucyl-leucyl-norlucinal, caused a rapid and near-complete loss of approximately 22-23-kDa ubiquitinated nucleoproteins, which we have identified as monoubiquitinated nucleosomal histones H2A and H2B by immunological and two-dimensional electrophoretic techniques. In human SKBr3 breast tumor cells, depletion of monoubiquitinated histones by the proteasome inhibitors coincided with the accumulation of high molecular weight ubiquitinated proteins in both nucleoprotein and cytosolic fractions and decreased unconjugated ubiquitin in the cytosol, without changes in the nonubiquitinated core histones. Unconjugated ubiquitin was not detected in isolated tumor cell nuclei. A similar loss in monoubiquitinated histones occurred in cells harboring a defective, temperature-sensitive mutation of the ubiquitin-activating E1 enzyme, after these cells were elevated from 33 degrees C to the non-permissive temperature of 39 degrees C. DNA replication and RNA transcription were decreased by the proteasome inhibitors most strongly after 90% of the ubiquitin had been removed from ubiquitinated histones H2A and H2B, suggesting a relationship between the nucleosomal histone ubiquitin status and the processing of genetic information. Interestingly, although both proteasome inhibitors caused a generalized decrease in methionine incorporation into proteins, they strongly induced the synthesis of the hsp72 and hsp90 stress proteins. Finally, treating cells with heat-shock at 43 degrees C, with stress response-provoking chemicals or with several other proteasome inhibitors caused ubiquitinated proteins to accumulate, depleted free ubiquitin, and concomitantly decreased nucleosomal monoubiquitinated histones. These results suggest that deubiquitination of nucleosomal histones H2A and H2B may play a previously unrecognized role in the cellular stress response, as well as in the processing of chromatin, and emphasize the important role of the proteasome in cellular homeostasis.

Previous studies suggest that dihydrofolate reductase (DHFR) regulates its own translation. Moreover, intracellular levels of DHFR protein increase following exposure to the antifolate methotrexate (MTX), suggesting that MTX may release the translational inhibition mediated by DHFR [Chu et al. (1993) Biochemistry 32,4756-4760; Ercikan et al. (1993) Adv. Exp. Med. Biol. 338, 537-540]. To further investigate the role of DHFR in translational autoregulation, we have considered the possibility that DHFR directly contacts its cognate mRNA. Binding studies using a series of truncated DHFR mRNAs as probes localized the DHFR/RNA interaction to a 100-base-pair region containing two putative stem-loop structures; initial studies indicated that both of these loop structures are involved in protein binding. Moreover, the binding of MTX to DHFR prevents interaction of the protein with its cognate mRNA, thereby relieving translational autoregulation.

Bryonia dioica (Cucurbitaceae family) produces at least two type I ribosome-inactivating proteins, bryodin 1 (BD1) and bryodin 2 (BD2). A cDNA sequence encoding BD1 was isolated from B. dioica leaf mRNA using degenerative oligonucleotides and codes for a 22 amino acid signal peptide followed by a protein of 267 residues. Expression of two recombinant BD1 (rBD1) forms in Escherichia coli yielded proteins of 267 (to the natural stop codon) and 247 amino acids (to the putative cleavage site yielding the mature protein) that had identical protein synthesis inhibition activity as compared to native BD1. The substitution of Lys for Glu at position 189 near the active site reduced the ability of rBD1 to inhibit protein synthesis by 10-fold. Toxicologic analysis showed that rBD1 was well tolerated in rodents with LD50 values of 40 mg/kg in mice and >25 mg/kg in rats. A crystal of mature rBD1 protein was used to collect X-ray diffraction data to 2.1 A resolution. The rBD1 crystal structure was solved and showed extensive homology with other type I RIPs and A chains of type II RIPs. The studies described here demonstrate that rBD1 retains full biologic activity and serve as a guide for using this potent, yet nontoxic, RIP in the construction of single-chain immunotoxin fusion proteins.

A eukaryotic translation initiation factor 2 (eIF-2)-associated 67 kDa glycoprotein (p67) protects the eIF-2 alpha-subunit from inhibitory phosphorylation by eIF-2 kinases, and this promotes protein synthesis in the presence of active eIF-2 alpha kinases in vitro [Ray, M. K., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 539-543]. We have now examined the effect of overexpression of this cellular eIF-2 kinase inhibitor in an in vivo system using transiently transfected COS-l cells. In this system, coexpression of genes that inhibit PKR activity restores translation of plasmid-derived mRNA. We now report the following. (1) Transient transfection of COS-1 cells with a p67 expression vector increased p67 synthesis by 20-fold over endogenous levels in the isolated subpopulation of transfected cells. (2) Cotransfection of p67 cDNA increased translation of plasmid-derived mRNAs. (3) Overexpression of p67 reduced phosphorylation of coexpressed eIF-2 alpha. (4) p67 synthesis was inhibited by cotransfection with an eIF-2 alpha mutant S51D, a mutant that mimics phosphorylated eIF-2 alpha, indicating that p67 cannot bypass translational inhibition mediated by phosphorylation of the eIF-2 alpha-subunit. These results show that the cellular protein p67 can reverse PKR-mediated translational inhibition in intact cells.

A protein synthesis cell-free system capable of performing with similar efficiencies in different ionic conditions has been developed for the halotolerant marine bacterium Vibrio costicola. The system has been used to test the effect of ionic strength on the interference produced by thirty translation inhibitors with different structural, functional, and domain specificities. In general, at high ionic strengths, the inhibition of protein synthesis produced by polycationic antibiotics like the aminoglycosides is much less pronounced than the inhibition obtained at low ionic strengths, while non-aminoglycosidic antibiotics show similar inhibitory activities at both high and low ionic conditions. These results strongly suggest that competition between polycationic antibiotics and cations at high concentrations in the media is responsible for the lack of inhibition by aminoglycoside antibiotics at high ionic strengths, rather than a lack of binding sites.

The heme-regulated inhibitor (HRI) of protein synthesis becomes activated in rabbit reticulocyte lysates in response to a variety of conditions including heme-deficiency, addition of oxidants, and heat shock. Activated HRI inhibits translation by catalyzing the phosphorylation of the alpha-subunit of eukaryotic initiation factor eIF-2. The molecular nature of the "signal" that leads to the activation of HRI in response to heat shock has not been characterized. We have recently reported that HRI interacts with the 90- and 70-kDa heat shock proteins (hsp) and a 56-kDa protein in hemin-supplemented lysates [Matts, R.L., Xu, Z., Pal, J.K., & Chen, J.-J. (1992) J. Biol. Chem. 267m 18160-18167]. In this report, we demonstrate that addition of denatured proteins, bovine serum albumin (BSA), beta-lactoglobulin, or alpha-lactalbumin, but not the addition of the native proteins, inhibits protein synthesis in hemin-supplemented reticulocyte lysates. The inhibition was reversed upon the addition of 10 mM cAMP or purified eIF-2B, classical criteria for HRI-mediated translational inhibition. Denatured BSA, but not native BSA, stimulated the phosphorylation of the alpha-subunit of eIF-2. This stimulation of eIF-2 alpha phosphorylation was inhibited by a monoclonal antibody to HRI, confirming that denatured BSA was causing the activation of HRI. The concentration of denatured BSA required to inhibit protein synthesis by 50% correlated with the levels of hsp70 present in each lysate preparation. Lysate hsp70 co-immunoadsorbed with denatured BSA, but not with not with native BSA. Hsp70 was co-adsorbed with HRI from lysate in the presence of native BSA, but not in the presence of denatured BSA.(ABSTRACT TRUNCATED AT 250 WORDS)

A mammalian cell infected with a human rhinovirus or enterovirus has a much reduced capability to translate capped mRNAs (the host cell shutoff), while still allowing translation of uncapped viral RNA. Biochemical and genetic evidence suggests that the viral proteinase 2A induces cleavage of the eukaryotic initiation factor (eIF) 4 gamma (also known as p220) component of eIF-4 (formerly called eIF-4F). However, neither the mechanism underlying the specific proteolysis of eIF-4 gamma nor the influence of this cleavage on the translation of capped mRNAs has been clarified. Such studies have been hampered by a lack of large quantities of a purified 2A proteinase. Therefore, the mature proteinases 2A of human rhinovirus 2 and coxsackievirus B4 were expressed in soluble form in Escherichia coli. A four-step purification protocol was developed; 1 mg of highly purified 2A proteinase per gram wet weight of E. coli was obtained. Both enzymes cleaved directly eIF-4 gamma as part of the purified eIF-4 complex. Addition of HRV2 2A proteinase to HeLa cell cytoplasmic translation extracts resulted in eIF-4 gamma cleavage and drastically reduced the translation of capped mRNA; addition of purified eIF-4 restored translation to the initial level. However, translation of a reporter gene driven by the 5'-untranslated region of human rhinovirus 2 was translated 2-3-fold more efficiently in the presence of HRV2 2A proteinase.

Transferrin, the transferrin receptor, and ferritin are integral to the body's management of iron, an element required for life but highly toxic when present in excess. The transferrin receptor and ferritin are regulated posttranscriptionally by iron: the transferrin receptor by mRNA stability and ferritin by mRNA translation. Results described here indicate that transferrin, like ferritin, is regulated by iron at the level of translation. Chimeric genes introduced into the mouse genome were composed of the human transferrin 5' regulatory region fused to the chloramphenicol acetyl transferase (CAT) reporter gene. Iron administration to transgenic mice resulted in a significant decrease of transferrin-directed CAT enzyme activity and CAT protein in liver, but no significant decrease in human transferrin-CAT mRNA levels. Binding of specific RNA iron regulatory elements by proteins in cytoplasmic extracts have been shown to regulate ferritin and transferrin receptor synthesis. Similar results have been obtained with transferrin mRNA. A decreased binding of human transferrin 5'-untranslated region RNA by factors in cytoplasmic extracts of livers from mice receiving iron was found when compared to extracts from control mice. A human transferrin RNA-protein complex migrated electrophoretically with the same mobility as a ferritin iron responsive element RNA-iron responsive element binding protein complex. The ferritin iron responsive element RNA also competed with the human transferrin 5'-untranslated region RNA-protein complexes formed and vice versa. Therefore, iron modulation of human transferrin may share a factor common or similar to that observed in ferritin and transferrin receptor iron modulation.

The messenger RNA rat alpha2u globulin has been identified and quantitated in a cell-free translational system derived from Krebs II ascites cells. Hepatic tissue of the mature male rats which normally produce alpha2u globulin was also found to contain a high level of alpha2u mRNA. Approximately 1.6 per cent of all poly(A) containing RNA of the adult male rat liver could be accounted for alpha2u messenger activity. Female rats do not produce alpha2u globulin and no alpha2u mRNA activity could be detected in the poly(A) containing RNA fraction obtained from the livers of these animals. However, androgen treatment to spayed female rats was found to induce the parallel appearance to both alpha2u globulin and its corresponding mRNA. Both hypophysectomy and adrenalectomy which are known to reduce the level of alpha2u globulin in the urine of male rats were found also to reduce the hepatic level of alpha2u mRNA. The results indicate that hormonal control of alpha2u globulin synthesis in rat liver is achieved primarily through regulation of its translatable mRNA level and that more than one hormone may participate in this regulation.

The effects of 5-fluorocytidine on ribosomal RNA maturation and structure in Novikoff hepatoma cells were investigated. Like other nucleic acid base analogues that are incorporated into RNA, this compound inhibits maturation of the 45S ribosomal RNA precursor. The 45S RNA precursor produced in the presence of 5-fluorocytidine has an abnormal electrophoretic mobility compared with that of the control precursor under nondenaturing conditions, but the two have identical mobilities under denaturing conditions. Under the conditions of these experiments, 5-fluorocytidine inhibited cellular protein synthesis only slightly, whereas equimolar concentrations of 5-azacytidine resulted in nearly 75% inhibition of this process. Despite this difference in the effects of the two analogues as well as the greater chemical lability of the 5-azacytidine, their effects on ribosomal RNA maturation are identical.

It has been known that insulin raises the rate of incorporation of [3H]leucine into the total protein of hormone-deficient chick embryo fibroblasts by approximately 1.5-fold. The elevation is not dependent upon the production of new messenger ribonucleic acids (mRNAs). Evidence is now presented in support of the following points: the greater labelling is due to more rapid polypeptide synthesis, not to an increase in the specific activity of leucyl-tRNA; the enhanced synthesis derives largely or entirely from a speeding up of the process of initiation, rather than that of elongation or termination; and the 1.5-fold stimulation is due to the elevated rates of formation of at least many of the fibroblast proteins. The hormone was shown before to stimulate posttranscriptionally and highly preferentially for formation of ribosomal proteins in the resting chick embryo cells. The question has been asked here whether insulin increases the production of total cell ribosomal protein by chemically altering preformed mRNAs. Results obtained by translating messages from deprived and hormone-treated cells in wheat germ and reticulocyte preparations do not support a mechanism involving covalent modification of preformed mRNAs. The observations, coupled with those previously made with inhibitors of translation, lead us to suggest that insulin stimulates protein synthesis in the resting chick embryo cells by activating limiting components of the initiation system. The effects of the hormone are greatest with messages, such as those for the ribosomal proteins, that have low affinities for the limiting initiation components.

Rat pituitary RNA was translated in the wheat germ system. Preprolactin messenger RNA activity was estimated by adsorption of cell-free products to solid phase antiprolactin. When male rats were injected for 4 days with estradiol-17beta, pituitary preprolactin mRNA activity was increased 2.5- to 3.0-fold over controls. This increase was evident when either total RNA, poly(adenylic acid) RNA, or polysomal RNA was translated in the cell-free system. In male rats receiving daily injections of estradiol-17beta, preprolactin mRNA activity was increased to an apparent maximum of 300% of controls after 7 days of treatment. Our data also indicate that estradiol increases preprolactin mRNA activity per microgram of RNA as well as the pituitary content of RNA. After estradiol treatment was discontinued, preprolactin mRNA activity declined to 50% of the maximum stimulation after approximately 2 days. In ovariectomized retired breeder female rats, a 5-fold increase in preprolactin activity over ovariectomized controls was obtained. In other studies, a 2-fold increase in preprolactin mRNA activity was obtained in male rats 24 h after a single injection of pimozide, a dopamine blocking drug.

The modes of action of insulin and of inhibitors of protein synthesis on the degradation of labeled cellular proteins have been studied in cultured hepatoma (HTC) cells. Protein breakdown is accelerated upon the deprivation of serum (normally present in the culture medium), and this enhancement is inhibited by either insulin or cycloheximide. An exception is a limited class of rapidly turning over cellular proteins, the degradation of which is not influenced by insulin or cycloheximide. Alternative hypotheses to explain the relationship of protein synthesis to the regulation of protein breakdown, viz., control by the levels of precursors of protein synthesis, regulation by the state of the ribosome cycle, or requirement for a product of protein synthesis, have been examined. Protein breakdown was not influenced by amino acid deprivation, and measurements of valyl-tRNA levels in HTC cells subjected to various experimental conditions showed no correlation between the levels of charged tRNAVal and the rates of protein degradation. Three different inhibitors of protein synthesis (puromycin, pactamycin, and cycloheximide) suppressed enhanced protein breakdown in a similar fashion. A direct relationship was found between the respective potencies of these drugs to inhibit protein synthesis and to block enhanced protein breakdown. When cycloheximide and insulin were added following a prior incubation of HTC cells in a serum-free medium, protein breakdown was maximally suppressed within 15-30 min. Actinomycin D inhibited protein breakdown only after a time lag of about 90 min. It is suggested that the regulation of protein breakdown in hepatoma cells requires the continuous formation of a product of protein synthesis, in a manner analogous to the mode of the control of this process in bacteria.

Inhibitors of RNA synthesis such as actinomycin D, 2-mercapto-1-(beta-4-pyridylethyl)benzimidazole, and cordycepin progressively inhibit the initiation of protein synthesis in intact nucleated mammalian cells independent of their effect on mRNA synthesis. The mechanism of this effect is unknown. The activity of cell-free lysates is not directly affected by these inhibitors, suggesting that their effect is indirect and requires an intact cell. However, lysates prepared from L-cells or CHO cells treated with the inhibitors do exhibit a decrease in initiation activity corresponding in magnitude to the effect seen in intact cells. Mixing experiments with lysates isolated from untreated or treated cells provide no evidence for a translational inhibitor. However, experiments analyzing the incorporation of [35S]methionine and [35S]Met-tRNAf into initiation complexes showed that while the level of labeled 40S initiation complex in lysates from treated cells was the same or higher than in control lysates, the rate or efficiency of formation of the 80S initiation complex was inhibited. These results imply that the transcriptional inhibitors do not affect the level or charging of the initiation tRNAMet, the activity of the eIF-2 initiation factor needed for ternary complex formation, and the availability of active 40S ribosomal subunits. Thus, this site of action is different from that observed in other translational control systems such as the hemin response in reticulocytes and the interferon-induced translation inhibition in virally infected cells. This effect may reflect the cell's coordination of nuclear transcription and cytoplasmic translation.

Ribosomal small subunits from canine pancreas were used to survey the effects of potassium chloride in the concentration range from 0.4 to 1.25 M. When combined with 60S particles, the treated 40S subunits showed no significant change in phenylalanine incorporating activity until exposure to 0.95 M KCl. Decreases in protein content of the subunits were observed at high ionic strengths. Attempts to separate dissociated ribosomal protein and the remaining core particles treated with 1.25 M KCl by centrifugation of the salt-treated particles though a 40% sucrose cushion led to the observation that ribosomal subparticles isolated in this manner retained full phenylalanine incorporating activity, whereas centrifugation through other solutions resulted in inactive or less active particles. Experiments were performed to elucidate the mechanism by which the 40% sucrose cushion was stabilizing the high salt treated 40S subunits. Two-dimensional gel electrophoresis of the proteins of the various particles isolated in the study was performed. The active 40S particles contained 23-31 protein spots. The isolation of fully active 40S subunits with fewer proteins than previously reported should simplify elucidating their role in the function of the small subunit.

A cell-free protein biosynthesizing system prepared from Escherichia coli CF300 was found to synthesize E. coli tryptophan synthase alpha subunit in a time-dependent manner when programmed with pBN69 plasmid DNA. This plasmid contains the trp promoter from Serratia marcescens adjacent to the coding region of E. coli tryptophan synthase alpha protein [Nichols, B.P., & Yanofsky, C. (1983) Methods Enzymol. 101, 155-164]. The synthesized tryptophan synthase alpha subunit was found to be indistinguishable from authentic alpha subunit protein when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and to have the same specific activity for catalyzing the conversion of indole----L-tryptophan by tryptophan synthase beta 2 subunit, as well as the conversion of indole + glyceraldehyde 3-phosphate to indole-3-glycerol phosphate. In the absence of exogenously added phenylalanine, admixture of E. coli phenylalanyl-tRNAPhe to the protein biosynthesizing system stimulated the production of functional alpha protein; the analogous result was obtained when valine was replaced by E. coli valyl-tRNAVal. The ability of a misacylated tRNA to participate in alpha protein synthesis in this system was established by the use of E. coli phenylalanyl-tRNAVal in the absence of added valine. Protein biosynthesis proceeded normally and gave a product having the approximate molecular weight of tryptophan synthase alpha subunit; as expected, this polypeptide lacked catalytic activity.

DNA complementary to purified ovalbumin messenger RNA (cDNA ov) was synthesized in vitro using RNA-directed DNA olymerase from avian myeloblastosis virus. This cDNAov was then employed in hybridization assays to determine the effect of estrogen on the number of ovalbumin mRNA (MRNAov) molecules per tubular gland cell of the chick oviduct. The changes in mRNAov were measured in immature chicks during primary stimulation, after hormone withdrawal and again following secondary stimulation of the chick oviduct with estrogen. The number of mRNAov per tubular gland cell was also determined for egg-laving hen. Daily estrogen administration to the immature chick resulted in growth of the oviduct, differentiation of epithelial cells to tubular glands, and a corresponding increase in the concentration of mRNAov in the tubular gland cell from essentially zero before estrogen administration to 48,000 molecules per cell after 18 days of estrogen treatment. Upon withdrawal of estrogen from the chick, the mRNAov concentration decreased to a level of 0-10 molecules/tubular gland cell after 12 days. Readministration of a single dose of estrogen to these chicks resulted in a dramatic and rapid increase in the concentration of mRNAov. Within 30 min, the mRNAov concentration approximately doubled and by 29 hr the tubular gland cell concentration had reached 17,000 molecules. The initial transcription rate for the ovalbumin gene was 12 mRNAov molecules/min. With these data, we have calculated that the half-life of the ovalbumin messinger RNA should be on the order of 40-60 hr and that the steady-state concentration of mRNAov per tubular gland cell was 50,000 molecules. Similarly, each messenger RNA molecule was translated approximately 50,000 times during its lifetime in order to effect the necessary quantity of ovalbumin required for egg production. These data substantiate the hypothisis that estrogen exerts its primary action at the level of transcription to effect the synthesis of nascene mRNA molecules which in turn code for synthesis of hormone-induced proteins.

A homologous in vitro method was developed from Tetrahymena for ribosomal A-site binding of aminoacyl-tRNA to poly(uridylic acid)-programmed ribosomes with very low error frequency. The reaction mixture pH was the crucial factor in the stable A-site association of aminoacyl-tRNA with high fidelity. At a pH greater than 7.1, endogenous activity translocated A-site-bound aminoacyl-tRNA to the P site. If translocation was allowed to occur, a near-cognate amino-acyl-tRNA, Leu-tRNA, could stably bind to the ribosome by translocation to the ribosomal P site. Near-cognate aminoacyl-tRNA did not stably bind to either site when translocation was blocked. Misreading antibiotics stimulated the stable association of near-cognate aminoacyl-tRNA to the ribosomal A site, thereby increasing the error frequency by several orders of magnitude. Ribosome binding of total aminoacyl-tRNA near equilibrium was not inhibited by misreading antibiotics; however, initial rate kinetics of the binding reaction were dramatically altered such that a 6-fold rate increase was observed with paromomycin or hygromycin B. The rate increase was evident with both cognate and near-cognate aminoacyl-tRNAs. Several antibiotics were tested for misreading potency by the ribosome binding method. We found gentamicin G418 greater than paromomycin greater than neomycin greater than hygromycin B greater than streptomycin in the potentiation of misreading. Tetracycline group antibiotics effectively inhibited A-site aminoacyl-tRNA binding without promoting misreading.

When the RNA from both the nucleus and polysomes of germinated cotton seeds were fractionated on poly(U)-cellulose columns it was found that both contained poly(A)-RNA. In ungerminated seeds most of the poly(A)-RNA is located in the nucleus of the cell. 3'-Deoxyadenosine was found to inhibit the synthesis of both nuclear and polysomal poly(A) by 75% in germinated seeds, but had little or no effect on protein synthesis during the first 6 hr of germination. After this time 3'-deoxyadenosine had a pronounced inhibitory effect on protein synthesis presumably due to the inhibition of poly(A) addition to newly synthesized mRNA.

The 70S ribosomes can select the proper initiator tRNA between Met-tRNAfMet and fMet-tRNAfMet. Experiments on binding and on formation of aminoacylpuromycin, as a function of magnesium, potassium, or initiation factors, suggest a two-state equilibrium for 70S particles, involving a minor, active conformation and a major one which is not readily active. The formyl group would act as a specific trigger to select the active conformation. Experimental results are interpreted following this simple model and equilibrium parameters, together with kinetic constants of the peptidyltransferase activity, are presented.

Lysates of L cells infected for 4 h with vesicular stomatitis virus were inhibited in their in vitro translational activity to about the same extent as protein synthesis was inhibited in vivo in infected L cells. Inhibition of translation occurred at the level of the ribosome as determined by reciprocal cross-reconstitution studies with polyribosomes and postribosomal supernatant fractions isolated from virus-infected and mock-infected cells. Inhibition of protein synthesis in reconstituted lysates of virus-infected cells was found to be at the level of initiation of translation as evidenced by reduction in incorporation into acid-precipitable proteins of formylatable [35S]methionine. Ribosomes from virus-infected and mock-infected cells were exposed to 0.5 M KCl and fractionated by centrifugation into salt-washed polyribosomes and supernatant fractions containing ribosome-associated proteins; reciprocal reconstitution of translational activity by a mixing of salt-washed polyribosomes and ribosome-associated proteins revealed that the defect in initiation of translation was in the ribosome-associated proteins released by salt wash from the infected-cell ribosomes. Differential ammonium sulfate precipitation of the supernatant ribosome-associated proteins from virus-infected and mock-infected cells indicated by reciprocal reconstitution studies that the defective ribosomal initiation factor(s) was (were) present primarily in the 0-40% ammonium sulfate fraction that is considered to contain primarily eIF-3 and eIF-4B. These results are similar to those found in earlier studies of defective initiation factors responsible for impaired protein synthesis in cells infected with plus-strand viruses quite different from the rhabdovirus studied in these experiments.

The extent of the inhibitory effect of ricin in polyphenylalanine synthesis by eukaryotic ribosomes is strongly dependent upon the concentration of ribosomes and the elongation factors EF 1 and EF2. Maximal inhibition by ricin, in this assay is observed when either ribosomes or the two elongation factors are added under limiting conditions, whereas ricin-treated ribosomes support protein synthesis at saturating concentrations of elongation factors and ribosomes. Similarly, the enzymatic binding of Phe-tRNA to ribosomes is drastically blocked in ricin-treated ribosomes when low EF 1 concentrations are added to the reaction mixture, but there is no inhibition when EF 1 is at saturating concentrations. Furthermore, formation of the complex EF 2-guanosine triphosphate-ribosome, using free ribosomes pretreated with ricin, is strongly inhibited at limiting concentrations of EF2, but is not affected at saturating concentrations of this factor. However, ricin does not inhibit the EF 2-dependent translocation of peptidyl-tRNA by polysomes, although the toxin is very active in preventing amino acid incorporation by polysomes. Our results suggest that the damaging effect of ricin on the ribosome causes a decreased affinity for both elongation factors EF 1 and EF 2. Thus, the toxin inhibits the enzymatic binding of aminoacyl-tRNA to ribosomes. The lack of inhibition of translocation by ricin suggests that the toxin cannot interact with ribosomes with substrate bound to the acceptor site. Essentially similar results are observed with ricin, abrin, ricin A chain, abrin A chain, and ricinus agglutinin A chain. A possible effect of the toxins on initiation and/or termination is further discussed.

A procedure is presented for the purification of alpha2u globulin, a protein synthesized in the liver, secreted into the serum, and excreted in the urine of male rats. The steady-state levels of alpha2u globulin in the serum and liver cytosol fraction of adult male rats have been determined using a radial immunodiffusion assay. A cell-free protein synthesizing system, easily prepared from commercial wheat germ, has been used to identify and quantitate the mRNA coding for alpha2u globulin. Poly(A)-containing RNA isolated from male rat liver directs the synthesis in the wheat germ translational system of a protein which is precipitated by rabbit anti-alpha2u globulin and migrates with authentic alpha2u globulin on sodium dodecyl sulfate-polyacrylamide gels. Poly(A)-containing RNA from the livers of female rats or from the kidneys of male rats, tissues which synthesize no alpha2u globulin, do not direct the synthesis of alpha2u globulin in the wheat germ system. Thyroidectomized male rats had no detectable alpha2u globulin in their sera or liver cytosols, and the livers from these thyroidectomized males were found to contain no translatable mRNA for alpha2u globulin, as measured in the wheat germ system. Administration of L-thyroxine or triiodo-L-thyronine to hyroidectomized males resulted in the synthesis of alpha2u globulin, as measured by increased levels of this protein in sera and liver cytosols. This increase in alpha2u globulin synthesis following thyroid hormone treatment was accompanied by a parallel increase in the functional level of the hepatic mRNA coding for this protein. Treatment of thyroidectomized males with a variety of androgens failed to stimulate alpha2u globulin synthesis, and no alpha2u globulin mRNA could be detected in the livers from these androgen-treated thyroidectomized males. These findings indicate that thyroid hormones influence alpha2u globulin synthesis in male rat liver by acting pretranslationally, possibly by modulating gene transcription, and rule out the possibility of an indirect androgen-mediated effect of thyroid hormones in modulating alpha2u globulin biosynthesis.

Two 5'-modified (2'-5')(A)4 oligomers with an increased resistance to phosphatase degradation were synthesized and evaluated for their ability to develop an antiviral response when introduced into intact cells by microinjection or by chemical conjugation to poly(L-lysine). The enzymatic synthesis of 5'-gamma-phosphorothioate and beta,gamma-difluoromethylene (2'-5')(A)4 from adenosine 5'-O-(3-thiotriphosphate) and adenosine beta,gamma-difluoromethylenetriphosphate by (2'-5')-oligoadenylate synthetase is described. The isolation and characterization of these (2'-5')(A)4 analogues were achieved by high-performance liquid chromatography. The structures of 5'-modified tetramers were corroborated by enzyme digestion. These two 5'-modified tetramers compete as efficiently as natural (2'-5')(A)4 for the binding of a radiolabeled (2'-5')(A)4 probe to ribonuclease (RNase) L. Nevertheless, at the opposite to 5'-gamma-phosphorothioate (2'-5')(A)4, beta,gamma-difluoromethylene (2'-5')(A)4 failed to induce an antiviral response after microinjection in HeLa cells. In addition, it behaves as an antagonist of RNase L as demonstrated by its ability to inhibit the antiviral properties of 5'-gamma-phosphorothioate (2'-5')(A)4 when both are microinjected in HeLa cells. The increased metabolic stability of 5'-gamma-phosphorothioate (2'-5')(A)4 as compared to that of (2'-5')(A)4 was first demonstrated in cell-free extracts and then confirmed in intact cells after introduction in the form of a conjugate to poly(L-lysine). Indeed, 5'-gamma-phosphorothioate (2'-5')(A)4-poly(L-lysine) conjugate induces protein synthesis inhibition and characteristic ribosomal RNA cleavages for longer times than unmodified (2'-5')(A)4-poly(L-lysine) in the same cell system.(ABSTRACT TRUNCATED AT 250 WORDS)

Total rough microsomes, isolated from the dog pancreas, were stripped of membranes-bound polysomes by treatment with either EDTA or puromycin and 0.5 M KCl. The stripped microsomal membranes were isolated relatively free from contamination, by using buoyant density centrifugation, and mRNA was isolated from both the membrane fraction and the released material. Depending on the method used to strip the rough microsomes, we found a variable but small percentage (3--15%) of the cellular poly(A)-containing mRNA attached to the microsomal membranes. Reextraction of isolated microsomal membranes with puromycin and 0.5 M KCl reduced the content of membrane-associated mRNA by approximately 50%, resulting in less than 2% of the total membrane-bound polysomal mRNA remaining associated with the microsomal membranes. The membrane-associated mRNA was characterized by translation in the wheat germ cell-free protein synthesizing system, and the products were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The translation products of the membrane-associated mRNA were identical with those from the total pancreas mRNA and also with those obtained by using mRNA isolated from material released directly from the rough microsomes.

A gentle and efficient method for selectively removing S1 from ribosomes was developed: the S1-free translation system prepared from such ribosomes is stimulated 10-20-fold (depending on the mRNA) by a stoichiometric amount of added purified S1. With this system, we examined the activity of mono- and di-N-ethylmaleimide derivatives of S1 in protein synthesis using synthetic and natural mRNAs and electrophoretic analysis of the translation products. The results show that ribosomes containing such modified S1's are functionally active although at a somewhat lower level (50-80% activity). Since treatment of S1 with N-ethylmaleimide abolishes the helix-destabilizing ability of S1, we conclude that this ability is not primarily responsible for S1's biological function. A new model for the role of S1 is proposed on the basis of the physical, structural, and RNA-binding properties of S1.