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Extracts of the conditionally-lethal mutant Escherichia coli N4316 are defective in a newly described translation factor, the rescue protein. We have analyzed the in vitro translation products of this mutant by gel electrophoresis during normal and arrested synthesis at the permissive and non-permissive temperatures. Translation programmed with MS2 bacteriophage RNA at the non-permissive temperature results in highly reduced synthesis of the coat protein with no detectable levels of the maturation and replicase products. Thus the relative number of copies of proteins synthesized by the ribosomes is altered in this mutant. In addition, there is mistranslation of the coat gene which results in the overproduction of the phage encoded no. 7 protein. Aberrant synthesis is also reflected in the increased read-through of termination codons during synthesis directed by phage RNAs harbouring amber mutations in the coat cistron. The rescue protein, purified from the parental strain, is able to complement the thermosensitive defect and restore proper synthesis. Biochemical characterization of the defect in the absence of rescue shows no detectable deficiency in the extent of initiation complex formation in reactions inhibited with sparsomycin. Peptidyltransferase is fully active as judged by the kinetics of formylmethionine-puromycin formation. However, rescue does exert an effect at the level of termination. In addition, the thermolability of the mutant can be reversed by dissociating 70S ribosomes into 30S and 50S subunits. Based on these and other observations, we propose tht rescue mediates a novel function in the association/dissociation of ribosomal subunits which is essential to the accuracy and efficiency of translation.

Tritiated elongation factors 1 and 2 (EF-1 and EF-2) were obtained from Krebs II ascites cells which had been grown in mice injected with radioactive amino acids. The highly purified factors were sufficiently radioactive to be used in a study of the interactions between ribosomes and elongation factors. The following results were obtained. 1. EF-1 binding to ribosomes requires the presence of a polynucleotide, an aminoacyl-tRNA specified by the latter and a guanosine nucleotide carrying three phosphate groups. The hydrolysis of the GTP molecule involved in the binding reaction leads to the immediate release of EF-1. If GTP is replaced by Guo-5'-P2-CH2-P the factor remains bound to the ribosome and can be detected by sucrose gradient centrifugation techniques. 2. Likewise EF-2 binding to ribosomes can only be detected in the presence of GUO-5'-P2-CH2-P. 3. The affinity of ribosomes for EF-2 appears to be higher than for EF-1: PREINCUBATION OF RIBOSOMES WITH EF-2 inhibits the subsequent attachment of EF-1 almost completely. EF-1 prebound to ribosomes in the presence of GUO-5'-P2-CH2-P, POLY(URIDYLIC ACID) AND Phe-tRNA-Phe is partially removed from the ribosomes together with Phe-tRNA during a second incubation with EF-2. 4. Although EF-2 binding to ribosomes precludes any stable association between ribosomes and EF-1 it does not prevent the insertion of aminoacyl-tRNA into the ribosomal A-site. The attachment of aminoacyl-tRNA under these conditions enhances the binding of EF-2 to the ribosome. 5. The antibiotic showdomycin strongly inhibits the attachment of EF-1 to ribosomes and to a lesser degree impairs the binding of EF-2. 6. A-site ribosomes display a strong preference for the attachment of EF-2 and bind EF-1 only very poorly. The reverse is true for P-site ribosomes which are good substrates for the binding of EF-1 and bind EF-2 less efficiently than A-site ribosomes. These results and a number of additional findings made in this and in previous studies are discussed in the general context of the structure and function of mammalian elongation factors 1 and 2.

1 An IF-M3-deficient ascites cell-free system is described. 2 An IF-M3 species of high molecular weight has been isolated and partially purified from a high-KCl ascites ribosomal wash fluid. 3 Translation of encephalomyocarditis virus RNA and globin mRNA is completely dependent upon the presence of the ascites IF-M3, while the factor is not required with either poly(A,U,G) or poly(U) as messengers. 4 The sparsomycin technique for the generation of short initiation peptides has been employed to demonstrate that IF-M3 acts at the level of chain initiation. 5 Rabbit reticulocyte IF-M3 and IF-E3, as well as rabbit liver IF-M3, can substitute for ascites IF-M3 in EMC-RNA and globin mRNA translation. 6 The translation of EMC-RNA in the ascites cell-free system is, therefore, dependent upon the presence of two discrete initiation factors, a high-molecular-weight species, IF-M3, and a smaller component, IFEMC.

In order to elucidate the mechanism of action of inhibitors that block the initiation of protein synthesis in mammalian systems, we have studied the following steps: (a) formation of the ternary complex Met-tRNAr-IF-E2-GTP, (b) binding of the initiator Met-tRNAf to the 40-S ribosomal subunit in the presence of initiation factors and dependent or not on the addition of mRNA, (c) formation of the initiation complex with 80-S ribosomes and (d) formation of the first peptide bond. Adrenochrome, aurintricarboxylic acid, polydextran sulphate, pyrochatechol violet and showdomycin block the formation of the ternary complex Met-tRNAf-IF-E2-GTP. Edeine A1, aurintricarboxylic acid and polydextran sulphate block the binding of the mRNA to the 40-S ribosomal subunit. Pactamycin induces the formation of stable smaller initiation complexes which are unable to go through the subsequent steps of initiation. Stimulation of the binding of the initiator Met-tRNAf to the 80-S ribosome in the presence of initiation factors is observed with sparsomycin and antibiotics of the sesquiterpene family (verrucarin A, trichodermin and trichothecin). However, these antibiotics block the reaction of the bound Met-tRNAf with puromycin. Narciclasine has no effect on the binding of the initiator to the ribosome but strongly blocks its reaction with puromycin. We have developed a simple technique to detect the Met-tRNAf-40-S-subunit-poly(A, G, U) initiation complexes by chromatography on Sepharose 6B columns. The requirements for the formation of such complexes measured by this technique and its comparison with the sucrose gradient centrifugation method are described.

The photochemical oxidation of the 50-S ribosomal subunit results in a rapid irreversible loss of peptidyl transferase activity. The first-order rate of inactivation occurring during the first forty minutes suggests that a single reactive group is being inactivated. The pH profile of inactivation exhibits a maximum at pH 7.5. Erythromycin at a low concentration (0.04 μmol) affords significant protection. Puromycin also exerts a protective effect but at higher concentrations. Chloramphenicol, sparsomycin and lincomycin did not exert a protective effect. The loss in catalytic activity was not accompanied by a loss in substrate binding affinity of the donor and acceptor substrates.

Extracts from interferon-treated Ehrlich ascites tumor cells differ in various biochemical characteristics from extracts from control cells. Thus, as reported earlier, double-stranded RNA (dsRNA) promotes the phosphorylation by ATP of at least two proteins in extracts from interferon-treated cells, but not, or to only a lesser extent, in extracts from control cells. Moreover reovirus mRNAs are degraded faster in reaction mixtures containing extracts from interferon-treated cells than in those containing extracts from control cells but only if the reaction mixtures are supplemented with dsRNA and ATP. The faster RNA degradation in extracts from interferon-treated cells is due to enhanced endonuclease action. We designated the agent(s) catalyzing this as endonucleaseINT. There is some enhancement of nuclease activity by dsRNA and ATP also in extracts from control cells. The extent of this enhancement is, however, much smaller than in extracts from interferon-treated cells. We report now that the promotion of mRNA cleavage in extracts from interferon-treated cells by dsRNA and ATP is apparently not a consequence of a possible impairment in the attachment of the mRNA to ribosomes since (a) the protein synthesis inhibitors (sparsomycin and edeine) do not affect the degradation and (b) dsRNA and ATP enhance RNA degradation also in the 200000 × g supernatant fraction from extracts of interferon-treated cells and this fraction is essentially free of ribosomes. (The ribosome concentration in our 200000 × g supernatant fraction is less than 1% of that in the 30000 × g extract.) GTP or the ATP analogs AMP(CH2)PP or AMP-P(CH2)P do not substitute for ATP and dsDNA or DNA · RNA hybrids do not substitute for dsRNA in activating endonucleaseINT. The optimal concentration of ATP in activating endonucleaseINT is 1 mM or higher. The addition of dsRNA and ATP to extracts from interferon-treated cells affects the degradation of some RNAs much more than that of others. Thus it promotes the degradation of reovirus mRNA from the large size class strongly, the degradation of those from the medium size class to an intermediate extent and that of those from the small size class the least. Furthermore it enhances the degradation of mRNA from either control or interferon-treated Ehrlich ascites cells more than that of Ehrlich ascites cell ribosomal RNA or mouse globin mRNA. Reovirus dsRNA is not cleaved by endonucleaseINT. The partially purified mouse interferon preparation (specific activity 8 × 108 NIH reference standard units/mg protein) used in several experiments was free of nuclease activity under our experimental conditions. Moreover the addition to extracts from control cells (or extracts from interferon-treated cells) of this interferon preparation together with dsRNA and ATP did not enhance nuclease activity.

Quantitative binding studies of [G-3H]anisomycin and [acetyl-14C]trichodermin to sensitive and resistant 80-S ribosomes from yeasts are described in this work. A single mutation, most probably affecting the ribosome peptidyl transferase centre, appears to have pleiotropic effects on the ribosome leading to resistance to trichodermin and anisomycin and to an increased sensitivity to sparsomycin. Resistance to trichodermin is due to a reduced affinity of ribosomes from the mutant for the antibiotic. Ribosomes from the sensitive strain (Y166) bind [acetyl-14C]trichodermin with a dissociation constant of 0.99 μM while those from the resistant one (TR1) bind [acetyl-14C]trichodermin with a dissociation constant of 15.4 μM. Similar results are obtained when the binding of [acetyl-14C]-trichodermin to Y166 and TR1 60-S subunits is studied. The mutant TR1 is also resistant to anisomycin. Although trichodermin and anisomycin bind to the ribosome at mutually exclusive sites, the higher affinity binding of [G-3H]anisomycin that is responsible for the inhibition of the peptidyl transferase center is practically identical for Y166 and TR1 ribosomes. Therefore, the mutation in the ribosome leading to resistance to trichodermin and anisomycin decreases the affinity for trichodermin but not for anisomycin. Trichodermin, trichothecin and fusarenon X inhibit the binding of [G-3H]anisomycin to TR1 ribosomes to a lower extent than to Y166 ribosomes, suggesting that the resistance of TR1 ribosomes to the effects of trichothecin and fusarenon X is caused by a decrease in the affinity of the ribosomes for these drugs, as was seen with trichodermin. On the other hand, verrucarin A inhibits [G-3H]anisomycin binding to Y166 and TR1 ribosomes to a similar extent and therefore its affinity for the ribosome does not appear to be affected by the mutation leading to resistance. Trichothecin, trichodermin and fusarenon X appear to have a common binding site on the 60-S ribosomal subunits, which overlaps or is closely linked to the binding sites of anisomycin and verrucarin A.

The quantitative interaction of [G-3H]gougerotin with Escherichia coli and Saccharomyces cerevisiae ribosomes is studied in this contribution. While E. coli ribosomes show an homogeneous single-site interaction with the antibiotic (dissociation constant = 1.5 μM), S. cerevisiae ribosomes are heterogeneous for [G-3H]gougerotin binding: there is a single-site interaction with dissociation constant = 0.9 μM in 28% of the ribosomes and another single binding site with dissociation constant = 12 μM in 58% of the ribosomes. Antibiotic interaction takes place in all cases with the larger ribosomal subunit. Gougerotin binding is affected by the nature of the ribosomal preparation. Thus, gougerotin has a much stronger affinity for E. coli washed ribosomes than for ribosomes reconstituted from their subunits. This difference is reduced when tRNAphe is prebound, in the presence of poly(U), to the reconstituted ribosomes. Gougerotin is also able to detect differences between the poly(U) · ribosome · Ac-Phe-tRNA complexes formed with either washed or reconstituted ribosomes. The effect of cations, pH, ethanol, temperature and inhibitors of peptide bond formation on [G-3H]gougerotin binding to E. coli and S. cerevisiae ribosomes has also been studied. Only those antibiotics that act on the peptidyl transferase centre of both prokaryotic and eukaryotic ribosomes (blasticidin S, sparsomycin and actinobolin) completely inhibit the binding of [G-3H]-gougerotin. In contrast, griseoviridin strongly enhances [G-3H] gougerotin binding to both types of ribosomes.

The isolation and properties of a novel N-formylmethionyl-tRNA hydrolase (hydrolase II) from Escherichia coli are described. This enzyme is difficult to detect in crude extracts; purification, however, unmasks the activity. Sedimentation and gel filtration parameters of this enzyme differ from those of the previously described peptidyl-tRNA hydrolase (hydrolase I), and preparations can be obtained where the two activities are free of each other. A mutant of hydrolase I has wild-type levels of hydrolase II. These data indicate that hydrolase II is a different enzyme, or an altogether different form of hydrolase 1. The bulk of the enzymic activity occurs in the ribosome-free cytoplasm; the remainder is found on intact or dissociated 70-S ribosomes. Purified preparations of hydrolase II analyzed by two-dimensional gel electrophoresis contain 2 protein bands. These 2 proteins do not coincide in electrophoretic mobility with any known ribosomal proteins. Analysis after mixing experiments verifies this conclusion. The purified enzyme (hydrolase II) is inhibited by ribosomes bearing bound N-formylmethionyl-tRNA. The inhibition is potentiated by sparsomycin and other antibiotics that block specifically peptide-bond synthesis. The relationship of this enzyme to other hydrolytic activities, including a newly described ribosome-dependent hydrolase, are discussed.

A rapid method for the quantitative and qualitative measurement of the initiation of protein synthesis in mammalian cell-free extracts is described. Use of the antibiotic sparsomycin, which inhibits chain elongation but has little effect on initiation, results in the accumulation of short initiation peptides during protein synthesis in vitro. The initiation peptides remain bound to tRNA but are not attacked by the putative aminopeptidase responsible for the removal of N-terminal methionine from nascent polypeptides. After electrophoresis and autoradiography such initiation peptides labelled with [35S] methionine yield a specific fingerprint for each site at which initiation has occurred. The optimal conditions for cell-free incubation, isolation and characterisation of initiation peptides are described.

1 The initiation of protein synthesis directed by the RNA from encephalomyocarditis virus in a cell-free system from Krebs II ascites cells has been investigated. 2 Ascites cell Met-tRNAMet donates all the internal methionyl residues of the viral-RNA-specified polypeptide, whereas little incorporation from Met-tRNAfMet is detected. 3 fMet-tRNAfMet, which is not normally present in the cytoplasm of eukaryotic cells, donates formylmethionine into polypeptide in response to the viral RNA to give a product which contains a single N-terminal sequence fMet-Ala-Thr-, and is identical to the normal cell-free polypeptide except for the N-terminal formylmethionyl residue. 4 Short-chain peptidyl-tRNA synthesised in vitro in response to encephalomyocarditis virus RNA in the presence of low concentrations of sparsomycin, which inhibits chain elongation but not initiation, contains the N-terminal sequence Met-Ala-Thr-. 5 This data supports the notion that unblocked Met-tRNAfMet is the universal initiator of protein synthesis in eukaryotes and donates a methionyl residue into the N-terminal position of all proteins. The initiating methionyl residue is in most cases subsequently removed from the nascent polypeptide, but this can be inhibited either by an N-terminal formyl group or by preventing the nascent polypeptide from growing long enough to be a substrate for the methionine aminopeptidase. 6 The data also shows that encephalomyocarditis virus RNA, which codes for at least nine stable proteins in vivo, contains only one initiation site which is expressed in vitro. The polypeptide coded for immediately after the initiation site is not virus-coat protein and is rapidly metabolised in vivo, suggesting that a lead-in polypeptide precedes the first meaningful protein sequence.