Protein Synthesis in Cell-Free Systems: an Effect of Interferon

The activity of ribosome and cell-sap fractions from interferon-treated and control chick embryo fibroblasts was compared in mixed chick-mouse and purely chick cell-free systems capable of the synthesis of viral polypeptide(s) in response to viral ribonucleic acid (RNA). Interferon treatment of cells did not affect the intrinsic amino acid incorporation activity of these systems or their response to polyuridylic acid. With encephalomyocarditis (EMC) virus RNA as messenger, however, a fraction of the ribosomes from interferon-treated cells appeared less active than parallel controls. The results obtained with the corresponding cell-sap fractions were variable. Although competition between endogenous and added messengers cannot be excluded in these systems, a reduced level of translation of EMC RNA with interferon-treated cell ribosomes was also suggested by the results of analyses of tryptic digests of the products formed in response to the RNA. In addition, these analyses showed that this reduced activity must reflect a reduction in the rate or frequency of translation rather than a decrease in the length of the EMC RNA translated, for the same polypeptides were synthesized in response to the RNA with material from interferon-treated and control cells. Interferon added directly to the cell-free system was without effect. Although suggestive, these results do not provide definitive evidence for or against the hypothesis that virus protein synthesis is inhibited at the translational level in the interferon-treated cell. Possible alternative interpretations of the data are discussed.     

Inhibition of protein synthesis by polypeptide antibiotics.. II. In vitro protein synthesis

Ennis, Herbert L. (St. Jude Children's Research Hospital, Memphis, Tenn.). Inhibition of protein synthesis by polypeptide antibiotics. II. In vitro protein synthesis. J. Bacteriol. 90:1109-1119. 1965.-This investigation has shown that the polypeptide antibiotics of the PA 114, vernamycin, and streptogramin complexes are potent inhibitors of the synthetic polynucleotide-stimulated incorporation of amino acids into hot trichloroacetic acid-insoluble peptide. The antibiotics inhibited the transfer of amino acid from aminoacyl-soluble ribonucleic acid (s-RNA) to peptide. The A component of the antibiotic complex was active alone in inhibiting in vitro protein synthesis, whereas the B fraction was totally inactive. However, the A component, when in combination with the B component, gave a greater degree of inhibition than that observed with the A fraction alone. On the other hand, the endogenous incorporation of amino acid was much less susceptible to inhibition than the incorporation of the corresponding amino acid in a system stimulated by synthetic polynucleotide. In addition, synthesis of polyphenylalanine stimulated by polyuridylic acid was inhibited to a greater extent when the antibiotics were added before the addition of polyuridylic acid to the reaction mixture than when the antibiotics were added after the polynucleotide had a chance to attach to the ribosomes. However, the antibiotics apparently did not inhibit the binding of C(14)-polyuridylic acid or C(14)-phenylalanyl-s-RNA to ribosomes. The antibiotics did not affect the normal release of nascent protein from ribosomes and did not disturb protein synthesis by causing misreading of the genetic code. The antibiotics bind irreversibly to the ribosome, or destroy the functional identity of the ribosome. The antibiotic action is apparently a result of the competition between antibiotic and messenger RNA for a functional site(s) on the ribosome.     

Evidence for a high proportion of inactive ribosomes in slow-growing yeast cells.

From the protein and RNA content of Saccharomyces cerevisiae growing in different media we calculate that ribosome efficiency is changed: incorporation of amino acids into protein decreases from 8.8 amino acids/s per ribosome in fast-growing cells (0.54 doubling/h) to 5.2 amino acids/s per ribosome in slow-growing cells (0.30 doubling/h). We could not detect significant protein turnover in either fast-or slow-growing cultures, so the lower ribosome efficiency does not seem to be an artifact caused by changes in unstable protein production at different growth rates. Nor is the lower ribosome efficiency due to slower migration of ribosomes along mRNA: the times required to complete polypeptides of known molecular weights are the same in slow-growing cells as those previously determined for fast-growing cells [Waldron, Jund & Lacroute (1974) FEBS Lett. 46, 11-16]. We therefore deduce that ribosome efficiency changes in yeast because the fraction of ribosomes engaged in protein synthesis falls (from 84% in fast-growing cells to 50% in slow-growing cells.     

Genomic RNA of mengovirus. VI. Translation of its two cistrons in lysates of interferon-treated cells.

The addition of low levels (40 ng/ml) of the synthetic double-stranded polyribonucleotide poly I:C to lysates of interferon-treated L-cells resulted in a strong inhibition (70 to 75%) of the in vitro translation of mengovirus RNA. Under these conditions, the rates of incorporation of [35S]methionine or formyl-[35S]methionine were depressed to a comparable extent. The sequences of mengovirus RNA recognized by ribosomes of interferon-treated cells at initiation of translation were compared with those present in initiation complexes formed by ribosomes of untreated controls. Fingerprint analysis revealed that the same sequences of mengovirus RNA were protected against nuclease attack by the 80S and the 40S initiation complexes formed in vitro in lysates of control or interferon-treated L-cells. Mengovirus RNA-coded proteins were labeled at their N-terminal end with formyl-[35S]methionine and digested to completion with trypsin. The resulting fragments were separated by high-voltage paper electrophoresis. Two different formyl-[35S]methionine-labeled N termini were resolved. Further analyses supported the notion that the two radioactive peaks originated in the initiation of translation at two different sites. This pattern did not change when mengovirus RNA was translated in lysates of interferon-treated cells.     

Protein synthesis by membrane-bound and free ribosomes of the developing rat cerebral cortex

1. Rates of RNA and protein synthesis were measured in rat cerebral-cortex slices, and compared with amino acid incorporation into protein by membrane-bound and free ribosomes from the same tissue, in the first 3 weeks of life. 2. A rapid age-dependent decline in the incorporation of labelled precursors into both RNA and protein was observed, which was more marked for amino acid incorporation into protein. 3. Although membrane-bound ribosomes comprise only a small fraction of total ribosomes, they were more active in incorporating amino acids into protein than were free ribosomes, especially immediately after birth. The decline in activity with age was more marked in the membrane-bound fraction than in free ribosomes. This loss of activity was largely independent of alterations in soluble factors or endogenous mRNA content and appeared to involve some alteration of the function of the ribosome itself, with relatively small alterations in the ratio of membrane-bound to free ribosomes. 4. Thyroidectomy, performed soon after birth, had no effect on the incorporation of radioactive precursors into RNA or protein by either slices or the cell-free preparations during the first 3-4 weeks of life.     

TRANSPORT OF VIRAL RNA IN KB CELLS INFECTED WITH ADENOVIRUS TYPE 2

Messenger RNA transport was studied in KB cells infected with the nuclear DNA virus adenovirus type 2. Addition of 0.04 µg/ml of actinomycin completes the inhibition of ribosome synthesis normally observed late after infection and apparently does not alter the pattern of viral RNA synthesis: Hybridization-inhibition experiments indicate that similar viral RNA sequences are transcribed in cells treated or untreated with actinomycin. The polysomal RNA synthesized during a 2 hr labeling period in the presence of actinomycin is at least 60% viral specific. Viral messenger RNA transport can occur in the absence of ribosome synthesis. When uridine-³H is added to a late-infected culture pretreated with actinomycin, viral RNA appears in the cytoplasm at 10 min, but the polysomes do not receive viral RNA-³H until 30 min have elapsed. Only 25% of the cytoplasmic viral RNA is in polyribosomes even when infected cells have been labeled for 150 min. The nonpolysomal viral RNA in cytoplasmic extracts sediments as a broad distribution from 10S to 80S and does not include a peak cosedimenting with 45S ribosome subunits. The newly formed messenger RNA that is ribosome associated is not equally distributed among the ribosomes; by comparison to polyribosomes, 74S ribosomes are deficient at least fivefold in receipt of new messenger RNA molecules.     

Studies of newly synthesized ribosomal ribonucleic acid of Escherichia coli

1. RNA synthesized by Escherichia coli during one-hundredth of the generation time contains two fractions distinguishable by hybridization with homologous DNA. One fraction, approximately 30% of the newly synthesized RNA, did not compete with ribosomal RNA, being apparently messenger RNA. The other fraction, approximately 70% of the newly made RNA, hybridized as ribosomal RNA. These values are comparable with previous estimates (McCarthy & Bolton, 1964; Pigott & Midgley, 1968). 2. Hybridization-competition experiments showed that the newly made RNA associated with 70s ribosomes and larger ribosome aggregates was a mixture of ribosomal RNA and messenger RNA, whereas that associated with nascent ribosomal subunits consisted exclusively of ribosomal RNA. This observation provides means by which newly synthesized ribosomal RNA can be isolated free from messenger RNA. 3. Newly made ribosomal RNA in nascent ribosomal subunits was sensitive to shear under conditions where ribosomal RNA in mature ribosomes was shear-resistant. Thus, when RNA was extracted from cells of E. coli disrupted by mechanical means, newly made ribosomal RNA appeared heterogeneous in size, sedimenting as a broad peak extending from 8s to 16s. 4. Newly synthesized ribosomal RNA in nascent ribosomal subunits was rapidly degraded in the presence of actinomycin D and during glucose starvation. 5. Newly synthesized ribosomal RNA stimulated amino acid incorporation in a system synthesizing protein in vitro to the same extent as the RNA which contained the messenger RNA fraction.     

Viral RNAs Associated with Ribosomes in Sindbis Virus-Infected HeLa Cells

Virus specific RNA ribosome complexes were isolated by sucrose density gradient centrifugation of cytoplasmic extracts from HeLa cells infected at 42 C with an RNA(+) mutant (ts2) of Sindbis virus. Viral RNA-ribosome complexes were accumulated by infected cells treated with sodium fluoride and cycloheximide. The RNA-ribosome complexes were characterized by (i) their sensitivity to the action of ribonuclease or ethylenediaminetetraacetic acid, (ii) their density in cesium chloride gradients, and (iii) presence of host ribosomes and viral RNAs. The viral RNAs were isolated and characterized. The results showed that two species of single-stranded RNAs (a 28s and 18 to 15s species) were associated with the complexes. Base composition analysis of the viral RNAs indicated that both species had a higher adenine content than the 42s or 26s forms of viral RNAs. The RNAs associated with the ribosome complexes were virus specific since they annealed with denatured double-stranded RNAs from the infected cells. Little or no 42S RNA was associated with the RNA-ribosome complexes. The results suggest that the 28s and 18 to 15s forms of RNAs may represent viral messenger RNAs.     

Single molecule imaging of the trans-translation entry process via anchoring of the tagged ribosome

Trans-translation is an eubacterial quality control system to rescue the stalled ribosome, in which multiple components such as transfer messenger RNA (tmRNA) and Small protein B (SmpB) are involved. However, how these molecules interact with ribosome remains elusive. Here, we report the single molecule analysis of the trans-translation process. We developed a new method to label the functional ribosome, in which a tag protein (the HaloTag protein of 297 amino acids) was fused to the 30S ribosomal protein S2 and covalently labelled with specific ligand (HaloTag ligand), resulting in the stable and specific labelling of ribosome. Ribosomes were anchored onto the glass surface using biotinylated derivative of the Cy3 HaloTag ligand (i.e. biotin-Cy3-ligand), and real-time interactions of Cy5-tmRNA/SmpB/EF-Tu ternary complexes with anchored ribosomes are observed as a model of the trans-translation entry. Statistical analysis revealed that Cy5-tmRNA/SmpB/EF-Tu ternary complexes bind to the anchored ribosome with the second-order rate constant of 2.6 × 10(6) (1/M/s) and tmRNAs undergo multi-modal pathway before release from ribosome. The methods presented here are also applicable to the analysis for general translation processes.     

Studies on the protein-synthesizing activity of the ribosomes of rat liver. The activity of free polysomes

1. The activities of microsome fractions from the liver of adult and 5-day-old rats for the incorporation of [(14)C]phenylalanine into protein were similar in the presence and absence of polyuridylic acid. 2. The activity of a light-microsome fraction from adult liver was greater than that of a heavy-microsome fraction, and the light-microsome fraction was also more markedly stimulated by the presence of polyuridylic acid. 3. The light-microsome fraction, when analysed by density-gradient centrifugation, contained a higher ratio of free ribosomes to bound ribosomes, whereas the reverse was true for the heavy-microsome fraction. Similar results were obtained for liver from adult and 5-day-old rats. 4. When the light-microsome fraction was incubated under conditions in which amino acid was incorporated into protein there was only a small increase in the ratio of free to bound ribosomes. When such a fraction was incubated with [(14)C]leucine and was then subjected to density-gradient centrifugation the fraction with the highest specific activity based on RNA had a density between that of the bound and free ribosomes. Treatment of the incubated fraction with ribonuclease shifted the radioactivity towards the free ribosome peak. These properties are consistent with the presence of active free polysomes. Such a component appeared also to be present when the heavy-microsome fraction was incubated under similar conditions. 5. The effect of the presence of polyuridylic acid on the incorporation of [(14)C]phenylalanine by the light-microsome fractions from liver of adult and 5-day-old rats was greatest in the region of the free ribosomes, but it is probable that some small polysomes containing polyuridylic acid are formed. 6. Polyuridylic acid also stimulated the bound ribosomes to a small extent when the heavy-microsome fraction from the liver of young rats was incubated with [(14)C]phenylalanine. 7. The results are discussed in terms of the various morphological constituents in liver now known to play a role in the synthesis of protein for export and for the internal activity of the cell.     

Mechanism of Interferon Action: Inhibition of Viral Messenger Ribonucleic Acid Translation in L-Cell Extracts

Encephalomyocarditis (EMC) virus ribonucleic acid (RNA) stimulated the incorporation of (14)C-amino acids into polypeptides in cell-free systems using preincubated S10 extracts from L cells. Incorporation was linear for over 2 hr. Analysis of the tryptic peptides derived from the polypeptide products formed in response to EMC RNA showed them to be virus specific. The major product, a polypeptide of 140,000 in molecular weight, migrated on sodium dodecyl sulfate-polyacrylamide gels with one of the virus-specific polypeptides present in EMC-infected cells. A minor component of molecular weight about 230,000 may correspond to the product of complete translation of the EMC virus genome. Little or no effect of interferon or vaccinia virus infection was observed in the preincubated, cell-free system. The EMC RNA-stimulated incorporation of (14)C-amino acids into polypeptides was not inhibited in extracts derived from L cells early in virus infection, from interferon-treated cells, or from cells subjected to both treatments. Interferon treatment did appear to have a slight inhibitory effect on chain elongation in this system. However, treatment of cells with highly purified interferon before virus infection caused a decrease of about 80% in the capacity of non-preincubated cell extracts to translate added EMC RNA. This effect did not extend to the translation of polyuridylic acid and could be reversed by preincubation of the extracts at 37 C for 20 min. The inhibition of translation was manifest at interferon concentrations as low as 5IU/ml, and in this respect closely paralleled the inhibition of virus growth. Inactivation of the antiviral activity of the interferon by heating or digestion with trypsin also abolished the effect on cell-free protein synthesis. The EMC-specific polypeptides formed in reduced amounts in extracts of interferon-treated vaccinia-infected cells were smaller than those formed in extracts of untreated, vaccinia-infected cells. Thus, inhibition of initiation or elongation of polypeptides, or both, can be demonstrated in cell-free systems employing non-preincubated extracts from interferon-treated, virus-infected cells. These results indicate that antiviral activity of interferon is directed against the translation of viral messenger RNA.     

Protein Synthesis in a Cell-Free Extract from Staphylococcus aureus

Cell-free Staphylococcus aureus extracts have been prepared which actively incorporate amino acids into protein. The requirements for amino acid incorporation of this preparation were strongly suggestive of de novo protein synthesis, since it showed an absolute requirement for ribosomes, 105,000 × g supernatant fluid, energy source, and magnesium ion. The stability of these extracts was greatly improved by use of dithiothreitol instead of mercaptoethanol as a sulfhydryl protecting reagent. Data were presented to show that the binding of aminoacyl-soluble ribonucleic acid to ribosomes did not require guanosine triphosphate and supernatant enzyme. The major characteristic which distinguishes this system from other cell-free systems is the much higher magnesium concentration required to maintain ribosomes intact and to obtain the maximal incorporation of amino acids. Addition of polyuridylic acid, polyadenylic acid, or polycytidylic acid caused about 60-fold, 30-fold, or 4-fold stimulation of the incorporation of phenylalanine, lysine, or proline, respectively. Studies by density gradient sedimentation indicated that radioactive polyuridylic acid or polyadenylic acid was associated with the monosomes. This complex can actively synthesize polypeptides. On the other hand, the nascent protein synthesized under the direction of endogenous messenger ribonucleic acid was associated with both polysomes and monosomes.     

Continued Expression of the Ribonucleic Acid Control Gene During Inhibition of Escherichia coli Ribonucleic Acid and Protein Synthesis

The effect of the ribonucleic acid (RNA) control (RC) gene on the biosynthesis of viral RNA has been examined in an RC(str) and an RC(rel) host infected with R17 RNA bacteriophage under conditions in which host RNA and protein synthesis were inhibited by the addition of rifampicin. Methionine and isoleucine starvation depressed viral RNA biosynthesis in an RC(str) host but not in an RC(rel) host. However, histidine starvation had little effect on viral RNA and protein synthesis in both RC(str) and RC(rel) cells, although it had a marked effect on host protein and RNA synthesis in an RC(str) host. Chloramphenicol relieved the effect of amino acid starvation on viral RNA synthesis in an RC(str) host. It is concluded that stringent control of viral RNA biosynthesis does not require the continued biosynthesis of the RC gene product (RNA or protein) and that a preformed RC gene product can regulate the biosynthesis of the exogenous RNA. It is suggested that the amino acid dependence of viral RNA biosynthesis is due to its obligatory coupling with the translation of the viral coat protein which lacks histidine. It may be inferred that the amino acid requirement of bacterial RNA is due to its coupling with the translation of a host-specific protein (other than the RC gene product) which requires a full complement of amino acids. Since chloramphenicol is known to permit ribosome movement in the absence of protein synthesis, it is suggested that ribosome movement along the nascent RNA chain is a sufficient condition for the continuation of RNA synthesis.     

RNA and ribosomal protein patterns during aerial spore germination in Streptomyces granaticolor

Disruption of the external sheath of Streptomyces granaticolor aerial spores and subsequent cultivation in a rich medium result in a synchronous germination. This method was used to analyze RNA and protein patterns during the germination. The germination process took place through a sequence of time-ordered events. RNA and protein synthesis started during the first 5 min and net DNA synthesis at 60-70 min of germination. Within the first 10 min of germination, synthesis of RNA was not sensitive to the inhibitory effect of rifamycin. During this period rRNA and other species including 4-5-S RNA were synthesized. Dormant spores contained populations of ribosomes or ribosomal precursors that were structurally and functionally defective. The ribosomal particles bound a sporulation pigment(s) of the melanine type. The ribosomal proteins complexed to the pigments formed insoluble aggregates which were easily removed from the ribosomes by one wash with 1 M NH4Cl. During the first 10 min of germination, pigment(s) were liberated from the complexes with the ribosomes and protein extracts of the washed ribosomes had essentially the same pattern as the extracts of ribosomes of vegetative cells. These structural alterations were accompanied by enhancement of the ribosome activities in polypeptide synthesis in vivo and in vitro. When the spores were incubated with a 14C-labelled amino acid mixture in the presence of rifamycin, only three proteins (GS1, GL1 and GS9) were identified to be radiolabelled in the extracts from the washed ribosomes. These experiments indicate that liberation of the sporulation pigment(s) from the complexes with ribosomal proteins and assembly of de novo synthesized proteins and proteins from a preexisting pool in the spore are involved in the reactivation of the ribosomes of dormant spores of S. granaticolor.     

Indiscriminate degradation of RNAs in interferon-treated, vaccinia virus-infected mouse L cells.

In this report we used Northern blot hybridization analysis to characterize the fate of several species of viral RNA transcribed from internal and terminal regions of vaccinia DNA in interferon-treated, infected mouse L cells grown in suspension. All species of viral RNAs were expressed but were reduced in amount. Larger-sized RNAs were reduced more than smaller-sized RNAs. This reduction appears to be related to the activation of the interferon-mediated double-stranded RNA-dependent 2-5A synthetase-endoribonuclease system, as the rRNA cleavage pattern characteristic of this system was observed early in infection and in cell extracts in response to exogenous 2-5A. Thus, in interferon-treated, vaccinia-infected mouse L cells in suspension, there is indiscriminate degradation of viral and cellular RNAs, and this RNA breakdown might play a role in the interferon-mediated inhibition of protein synthesis.     

A non-toxic pokeweed antiviral protein mutant inhibits pathogen infection via a novel salicylic acid-independent pathway

Pokeweed antiviral protein (PAP), a ribosome-inactivating protein isolated from Phytolacca americana, is characterized by its ability to depurinate the sarcin/ricin (S/R) loop of the large rRNA of prokaryotic and eukaryotic ribosomes. In this study, we present evidence that PAP is associated with ribosomes and depurinates tobacco ribosomes in vivo by removing more than one adenine and a guanine. A mutant of pokeweed antiviral protein, PAPn, which has a single amino acid substitution (G75D), did not bind ribosomes efficiently, indicating that Gly-75 in the N-terminal domain is critical for the binding of PAP to ribosomes. PAPn did not depurinate ribosomes and was non-toxic when expressed in transgenic tobacco plants. Unlike wild-type PAP and a C-terminal deletion mutant, transgenic plants expressing PAPn did not have elevated levels of acidic pathogenesis-related (PR) proteins. PAPn, like other forms of PAP, did not trigger production of salicylic acid (SA) in transgenic plants. Expression of the basic PR proteins, the wound-inducible protein kinase and protease inhibitor II, was induced in PAPn-expressing transgenic plants and these plants were resistant to viral and fungal infection. These results demonstrate that PAPn activates a particular SA-independent, stress-associated signal transduction pathway and confers pathogen resistance in the absence of ribosome binding, rRNA depurination and acidic PR protein production.     

Inhibition of Ribonucleic Acid Synthesis by Nalidixic Acid in Escherichia coli

The effect of low concentrations of nalidixic acid on ribonucleic acid (RNA) synthesis in Escherichia coli was examined. It was observed that RNA synthesis in exponentially growing cells was not significantly affected, in harmony with previous studies. However, RNA synthesis was markedly depressed by nalidixic acid during starvation for an amino acid or during chloramphenicol treatment. This effect was not caused by increased killing or inhibition of nucleoside triphosphate synthesis by nalidixic acid. The pattern of radioactive uracil incorporation into transfer RNA or ribosomes was not changed by the drug. The sensitivity of RNA synthesis to nalidixic acid in the absence of protein production may be useful in probing the amino acid control of RNA synthesis.     

Translation of Pseudomonas aeruginosa Bacteriophage PP7 RNA by a Cell-Free Amino Acid Incorporating System from Escherichia coli

We have compared the activities of the RNA genomes of Pseudomonas aeruginosa phage PP7 and coliphages Qbeta and f2 in a cell-free amino acid incorporating system derived from Escherichia coli. The rate of incorporation of [(14)C]leucine in the PP7 RNA-directed system is greater than in the systems directed by either Qbeta or f2 RNA. The response to changes in phage RNA concentrations is similar in all the systems, reaching a saturation level at 0.75 to 1.0 mg of RNA per ml of reaction mixture. Analysis of complete reaction mixtures of the PP7 RNA and of the Qbeta RNA systems by sucrose gradient centrifugation shows generally similar patterns for both RNAs. The principal differences are that in the PP7 system a slightly higher percentage of RNA forms ribosome complexes and that the polysomes are somewhat smaller. PP7 RNA is also degraded more extensively during the reaction than is Qbeta RNA. Analysis of the products of the reactions by acrylamide gel electrophoresis shows that PP7 coat protein is the only identifiable product of the PP7 RNA-directed system, suggesting that only the coat protein cistron is translated by E. coli ribosomes.     

Accumulation of 70S Monoribosomes in Escherichia coli After Energy Source Shift-Down

When Escherichia coli is shifted from glucose-minimal to succinate-minimal medium, a transient inhibition of protein synthesis and a time-dependent redistribution of ribosomes from polysomes to 70S monosomes occurs. These processes are reversed by a shift-up with glucose. In a lysate made from a mixture of log-phase and down-shifted cells, the 70S monosomes are derived solely from the down-shifted cells and are therefore not produced by polysome breakage during preparation. This conclusion is supported by the absence of nascent proteins from the 70S peak. The monosomes are not dissociated by NaCl or by a crude ribosome dissociation factor, so they behave as "complexed" rather than "free" particles. When down-shifted cells are incubated with rifampin to block ribonucleic acid (RNA) synthesis, the 70S monosomes disappear with a half-life of 15 min. When glucose is also added this half-life decreases to 3 min. The 70S particles are stable in the presence of rifampin when chloramphenicol is added to block protein synthesis. We interpret these data to mean that the existence of the 70S monosomes depends on the continued synthesis of messenger RNA and their conversion to free ribosomes (which dissociate under our conditions) is a result of their participation in protein synthesis. Finally, a significant fraction of the RNA labeled during a brief pulse of (3)H-uracil is found associated with the 70S peak. These results are consistent with the hypothesis that the 70S monosomes are initiation complexes of single ribosomes and messenger RNA, which do not initiate polypeptide synthesis during a shift-down.