Fate of mRNA following disaggregation of brain polysomes after administration of (+)-lysergic acid diethylamide in vivo

Intravenous injection of (+)-lysergic acid diethylamide into young rabbits induced a transient brain-specific disaggregation of polysomes to monosomes. Investigation of the fate of mRNA revealed that brain poly(A+)mRNA was conserved. In particular, mRNA coding for brain-specific S100 protein was not degraded, nor was it released into free ribonucleoprotein particles. Following the (+)-lysergic acid diethylamide-induced disaggregation of polysomes, mRNA shifted from polysomes and accumulated on monosomes. Formation of a blocked monosome complex, which contained intact mRNA and 40-S plus 60-S ribosomal subunits but lacked nascent peptide chains, suggested that (+)-lysergic acid diethylamide inhibited brain protein synthesis at a specific stage of late initiation or early elongation.     

Inhibition of protein synthesis in mouse myeloma cells by Ricinus communis toxin.

The mechanism of inhibition of protein synthesis in mouse myeloma cells by Ricinus communis toxin was studied. No significant disaggregation of polysomes into monosomes was detected in the toxin-treated cells. The activity of the polysomes isolated from the cells treated with the toxin in protein synthesis was remarkably lower than that of the untreated cells, while the activity of the supernatant enzyme fraction was retained. The ribosomes derived from the polysomes of the toxin-treated cells were inactive in poly(U)-dependent polyphenylalanine synthesis. The activity of ribosomes reconstituted by hybridizing subunits derived from the ribosomes of normal and toxin-treated cells were measured in poly(U)-dependent polyphenylalanine synthesis, and the 60 S subunit was revealed to be inactive. These results indicate that the target of action of the toxin towards intact cells is the 60 S ribosomal subunit.     

Messenger RNA affinity column fractionation of eukaryotic initiation factor and the translation of myosin messenger RNA.

Both myosin mRNA (26 S) and globin mRNA (9 S) have been bound to activated Sepharose 4B. The affinity of initiation factors derived from native 40 S ribosomal subunits from embryonic chick muscle for these messengers has been determined. Although both messengers bind the major components of the muscle factor preparation with the same affinity, some differences are noted in the minor components. There is an enrichment of components which bind myosin mRNA with a high affinity when the 15–18 S initiation factor complex is prepared from initiating 40 S ribosomal subunits found on myosin synthesizing polysomes rather than from total cellular factor preparations. The proteins which have a high binding affinity to myosin mRNA also have a discriminating effect when added to a wheat germ system containing myosin and globin mRNA. This is demonstrated by the fact that the synthesis of myosin heavy chain is specifically stimulated and the number of ribosomes found on myosin mRNA increase five to seven-fold; whereas neither the synthesis of globin nor the number of ribosomes associated with globin mRNA is increased. The components of an impure reticulocyte eukaryotic initiation factor 3 prepared in a similar manner as the muscle factor, do not bind myosin mRNA with the same high affinity, and these fractions separated on the myosin mRNA affinity column did not show a discriminatory effect. These results suggest that specific components of muscle 15–18 S initiation factor preparations have a higher binding affinity for myosin mRNA than globin mRNA and that these proteins may be those factors previously reported to be present which discriminate between mRNAs.     

The regulation of protein synthesis by translation control RNA

The mechanism by which translational control RNA (tcRNA) inhibits protein synthesis was investigated. In the presence of heme the inhibitory role of muscle tcRNA on hemoglobin synthesis was confirmed. Upon the addition of muscle tcRNA to a rabbit reticulocyte cell-free system the binding of [³²P]-globin mRNA to 40S ribosomal subunits and its subsequent incorporation into polysomes was inhibited. Furthermore, muscle tcRNA inhibits met-tRNA binding to polysomes and yet stimulates the formation of methionine-puromycin. These results suggest that muscle tcRNA blocks the binding of globin mRNA to ribosomes resulting in an abortive initiation complex that is, however, still capable of the methionine-puromycin reaction.     

Evidence that phosphorylation of eIF-2(alpha) prevents the eIF-2B-mediated dissociation of eIF-2 X GDP from the 60 S subunit of complete initiation complexes

Recent observations have indicated that eukaryotic initiation factor (eIF)-2 and GTP or GDP normally bind to 60 S ribosomal subunits in rabbit reticulocyte lysate and that when eIF-2 alpha is phosphorylated and polypeptide chain initiation is inhibited, eIF-2 X GDP accumulates on 60 S subunits due to impaired dissociation that is normally mediated by the reversing factor (eIF-2B). Current findings now indicate that inhibition due to phosphorylation of eIF-2 alpha is mediated, at least in part, by the inability to dissociate eIF-2 X GDP from the 60 S subunit of complete initiation complexes. At the onset of inhibition, there is an accumulation of Met-tRNA(f) and eIF-2 on the polysomes, despite a marked reduction in Met-tRNA(f) bound to 40 S subunits and Met-peptidyl-tRNA bound to the polysomes. This initial effect is not associated with the formation of "half-mers" (polysomes containing an extra unpaired 40 S subunit), and the 40 S X Met-tRNA(f) complexes, though reduced, still sediment at 43 S. When inhibition is maximal and the polysomes are largely disaggregated, there is an accumulation of 48 S complexes consisting of a 40 S subunit and Met-tRNA(f) bound to globin mRNA as well as small polysomal half-mers, such that residual protein synthesis occurs to about the same degree on "1 1/2"s and "2 1/2"s as on mono-, di-, and triribosomes. Exogenous eIF-2B increases protein synthesis on mono-, di-, and triribosomes and decreases that on half-mers. This is associated with reduced binding of Met-tRNA(f) and eIF-2 to ribosomal particles sedimenting at 80 S and greater and a shift from 48 S to 43 S complexes. These results suggest that eIF-2B must normally promote dissociation of eIF-2 X GDP from the 60 S subunit of complete initiation complexes before they can elongate but cannot when eIF-2 alpha is phosphorylated, resulting in the accumulation of these complexes, some of which dissociate into Met-tRNA(f) X 40 S X mRNA and 60 S X eIF-2 X GDP.     

Inhibition of protein synthesis by N-methyl-N-nitrosourea in vivo

1. The intraperitoneal injection of N-methyl-N-nitrosourea (100mg/kg) caused a partial inhibition of protein synthesis in several organs of the rat, the maximum effect occurring after 2-3h. 2. In the liver the inhibition of protein synthesis was paralleled by a marked disaggregation of polyribosomes and an increase in ribosome monomers and ribosomal subunits. No significant breakdown of polyribosomes was found in adult rat brains although N-methyl-N-nitrosourea inhibited cerebral and hepatic protein synthesis to a similar extent. In weanling rats N-methyl-N-nitrosourea caused a shift in the cerebral polyribosome profile similar to but less marked than that in rat liver. 3. Reaction of polyribosomal RNA with N-[(14)C]methyl-N-nitrosourea in vitro did not lead to a disaggregation of polyribosomes although the amounts of 7-methylguanine produced were up to twenty times higher than those found after administration of sublethal doses in vivo. 4. It was concluded that changes in the polyribosome profile induced by N-methyl-N-nitrosourea may reflect the mechanism of inhibition of protein synthesis rather than being a direct consequence of the methylation of polyribosomal mRNA.     

In vitro synthesis of RNA with “aggregate” enzyme,chromatin, and DNA from liver of methylazoxymethanol acetate-treated rats

“Aggregate” enzyme, chromatin and DNA preparations were isolated from livers of rats treated with the carcinogen, methylazoxymethanol (MAM) acetate. DNA template activity for RNA synthesis in vitro was unimpaired while the template activity of chromatin was slightly reduced. There was a marked inhibition of UTP incorporation into RNA, however, when the “aggregate” enzyme preparation was the source of both template and RNA polymerase. Circular dichroism analysis of the “aggregate” enzyme preparation indicated a change in conformation of the protein component. The results suggest that MAM acetate interacts with nuclear proteins and produces conformational changes which result in a decreased RNA synthesis.     

Preparation and characterization of a cell-free system from Chinese hamster ovary cells that translates natural messenger ribonucleic acid and analysis of intermediary reactions

A cell-free system from cultured Chinese hamster ovary cells has been developed, which translates endogenous mRNAs, exogenous natural mRNAs, and synthetic polynucleotide templates. The analysis of most of the reactions involved in initiation, elongation, and termination of protein synthesis can be carried out in this system. The postmitochondrial fraction, containing ribosomal 40 and 60 S subunits, 80 S ribosomes, polysomes, and cytosol proteins, incorporates amino acids into protein. The preparation is capable of recycling endogenous mRNA by initiating protein synthesis on polysomal mRNA, and of initiating protein synthesis on exogenous templates. When endogenous mRNA is degraded with micrococcal nuclease, polysomes are no longer evident and protein synthesis is markedly depended on added mRNA, ATP, GTP, and a nucleoside triphosphate-generating system. Amino acid incorporation is linear for over 2 h, polysomes containing nascent polypeptide chains are reformed and, with time, most of the protein synthesized is released into the media. Gel electrophoretic analysis of the product formed in response to globin mRNA indicates that most of the radioactivity migrates as a single peak, in the region corresponding to globin. Comparison of the electrophoretic pattern obtained from labeled Chinese hamster ovary cells with that from incubations of cell extract and Chinese hamster ovary mRNA indicates that essentially all of the polypeptides formed by the intact cell are synthesized by the cell-free system. Sucrose gradient centrifugation of incubations containing mRNA-depleted extract and [³⁵S]methionine, in the absence of added mRNA, is used to detect initiation intermediates in the formation of the [40 S Met-tRNA_f] complex and, with added natural mRNA plus cycloheximide, to detect intermediates in the formation of the 80 S initiation complex. Chain elongation reactions are measured by the incorporation of [³H]phenylalanine into polyphenylalanine in extracts supplemented with poly(U), or by the formation of nascent polypeptide chains on polysomes with natural mRNA. Chain termination is measured by analyzing the amount of radioactive protein released into the cytosol.     

Transfer to proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components

The data presented in this paper demonstrate that native small ribosomal subunits from reticulocytes (containing initiation factors) and large ribosomal subunits derived from free polysomes of reticulocytes by the puromycin-KCl procedures can function with stripped microsomes derived from dog pancreas rough microsomes in a protein-synthesizing system in vitro in response to added IgG light chain mRNA so as to segregate the translation product in a proteolysis- resistant space. No such segregation took place for the translation product of globin mRNA. In addition to their ability to segregate the translation product of a specific heterologous mRNA, native dog pancreas rough microsomes as well as derived stripped microsomes were able to proteolytically process the larger, primary translation product in an apparently correct manner, as evidenced by the identical mol wt of the segregated translation product and the authentic secreted light chain. Segregation as well as proteolytic processing by native and stripped microsomes occurred only during ongoing translation but not after completion of translation. Attempts to solubilize the proteolytic processing activity, presumably localized in the microsomal membrane by detergent treatment, and to achieve proteolytic processing of the completed light chain precursor protein failed. Taken together, these results establish unequivocally that the information for segregation of a translation product is encoded in the mRNA itself, not in the protein- synthesizing apparatus; this provides strong evidence in support of the signal hypothesis.     

Cross-linking of mRNA to initiation factor eIF-3, 24 kDa cap binding protein and ribosomal proteins S1, S3/3a, S6 and S11 within the 48S pre-initiation complex

Native small ribosomal subunits from rabbit reticulocytes contain all initiation factors necessary for the formation of the mRNA-containing 48S pre-initiation complex. The complex formed in the presence of Met-tRNAf and 125I-labelled globin mRNA was cross-linked with diepoxybutane, and the covalent mRNA-protein complexes were isolated under denaturating conditions. The proteins of the covalent complex were identified as the 110, 95 and 66/64 kDa subunits of eIF-3. In addition, the 24 kDa cap binding protein and the ribosomal proteins S1, S3/3a, S6 and S11 were found covalently linked to the mRNA. Ribosomal proteins S3/3a and S6 were also involved in the ribosomal mRNA-binding domain of reticulocyte polysomes.     

Inhibition of the initiation of hepatic protein synthesis during ethionine mediated ATP depletion in vivo: modification to ribosomal subunits, evidence of impaired ternary complex formation and a subcellular redistribution of eIF-2 alpha

Acute ethionine intoxication is known to induce a reversible hepatic injury in female rats by reducing the level of hepatic ATP. The injury indirectly impairs the initiation of hepatic protein synthesis, with resultant polysome disaggregation. Administration of adenine rapidly restores the ATP levels and protein synthesis. Analysis of liver polysome and ribosomal subunits reveals that polysome disaggregation occurs following 3 h of the intoxication, and reaggregation occurs following the administration of adenine. Inactive hepatic ribosomes accumulate as monomers and disomes when analysed by sucrose gradient sedimentation in low-salt buffers. High-salt buffers dissociate the inactive ribosomes into the component 40 S and 60 S subunits. The level of higher density, 1.48 g/cc, 40 S subunit increases during the inhibition of protein synthesis, while the lower density, 1.41 g/cc, 40 S subunit species does not change significantly. Hepatic microsomal and cytosolic extracts examined for their ability to support the formation of the ternary complex of eIF-2-GTP and [35S]Met-tRNAi demonstrate that during acute ethionine intoxication, ternary complex formation in the two extracts decrease 65% and 85%, respectively. These changes are coincident with polysome disaggregation. Administration of adenine to reverse the intoxication restores the ternary complex forming ability of the cytosolic extract, but does not affect the activity of the microsomal salt wash extracts. Mixing experiments indicate the accumulation of an inhibitor of ternary complex formation in the microsomal salt wash fraction. The application of quantitative western blotting demonstrates that the level of antigenic eIF-2 alpha in the microsomal salt wash extract increases 31% during the inhibition. These observations are consistent with the idea that the inhibition of the initiation of hepatic protein synthesis induced by ethionine is mediated by eIF-2 alpha phosphorylation. The latter results in an inhibition of ternary complex formation, redistribution of eIF-2 to the microsome fraction, polysomal disaggregation, and accumulation of inactive ribosomal subunits.     

Immunological detection of the messenger RNA cap-binding protein

The 24-kilodalton messenger RNA cap-binding protein (CBP) was purified from the rabbit reticulocyte postribosomal supernatant fraction using an affinity resin consisting of the p-aminophenyl gamma-ester of m7GTP coupled to Sepharose. The affinity-purified CBP was used to raise a goat antiserum. Anti-CBP antibodies were purified by adsorption to CBP coupled to either Controlled-Pore Glass or diazobenzyloxymethyl paper. The affinity-purified antibodies reacted specifically with only the 24-kilodalton polypeptide in whole reticulocyte lysate and in initiation factors prepared from the same source. During a conventional (nonaffinity) purification of CBP from a high salt extract of the ribosomal pellet, immunological reactivity paralleled the ability to reverse cap analogue inhibition of translation, indicating that the 24-kilodalton polypeptide present in the postribosomal supernatant fraction is immunologically cross-reactive with the CBP purified from ribosomes. Fractionation of whole reticulocyte lysate by sucrose gradient sedimentation followed by immunoblotting revealed that CBP was present in the supernatant fraction and the region of the gradient corresponding to ribosomal subunits but not in mono- or polysomes. The CBP to ribosome ratio was found to be approximately 0.02, assuming that the m7GTP-Sepharose retains all of the protein. This is considerably lower than that of other initiation factors and suggests that CBP may be the limiting polypeptide factor involved in the initiation of protein synthesis. The antibodies also inhibited the translation of a capped messenger RNA (globin). Inhibition of the translation of an uncapped RNA (satellite tobacco necrosis virus) was also observed, but to a lesser degree than with globin mRNA.     

Dual effects of the ricin A chain on protein synthesis in rabbit reticulocyte lysate. Inhibition of initiation and translocation

Ricin A chain caused inhibition of protein synthesis by reticulocyte lysate with concomitant depurination of 28S rRNA. The partial reaction(s) of protein synthesis inhibited was investigated by following the appearance of [35S]methionine from initiator [35S]Met-tRNA into 40S ribosomal subunits, 80S monosomes and polysomes. Ricin A chain caused an accumulation of [35S]Met in monosomes which did not enter polysomes. In these respects the effects of the ricin A chain resembled those of diphtheria toxin, an inhibitor of elongation-factor-2-catalyzed translocation. This is consistent with the previously proposed site of action of ricin as an inhibitor of elongation. However, the inhibitory effects of the ricin A chain and diphtheria toxin are not equivalent because we observed that the rate of formation of the 80S initiation complex was reduced approximately sixfold with the ricin A chain relative to diphtheria toxin. Analysis of methionine-containing peptides bound to 80S monosomes in ricin-A-chain-inhibited and diphtheria-toxin-inhibited lysates, programmed with globin mRNA, revealed a predominance of Met-Val, suggesting that the elongation cycle is inhibited at the translocation step. Translocation was also implicated as the step blocked in both the ricin-A-chain-inhibited and diphtheria-toxin-inhibited lysates, by the finding that nascent peptide chains were unreactive towards puromycin. It is concluded that ricin-A-chain-modified ribosomes are deficient in two protein synthesis partial reactions: the formation of the 80S initiation complex during initiation and the translocation step of the elongation cycle.     

The effect of non-permissive temperature on met-tRNA synthetase in Saccharomyces cerevisiae temperature-sensitive mutant ts 7-45

The effects of incubation of yeast spheroplasts at elevated temperature (40 degrees C) on a number of activities involved in protein biosynthesis have been examined in preparations obtained from wild-type cells (wt A364A ) and a temperature-sensitive mutant (ts 7-45) derived from it. With wild-type cells, preincubation of spheroplasts at the elevated temperature had little or no effect on the following: the ribosomal subunit-polysome pattern; the translation of exogenous natural mRNA in postpolysomal extracts devoid of endogenous mRNA; the translation of poly(U) in postpolysomal extracts; the incorporation of methionine into 40 S preinitiation and 80 S initiation complexes; the synthesis of Met-tRNA in postribosomal (cytosol) extracts; and the formation of eIF-2 X GTP X Met-tRNAf ternary complex in the cytosol. With temperature-sensitive spheroplasts that had not been preincubated at the elevated temperature, the concentration of free, native 40 S subunits appeared to be lower and that of 60 S subunits higher than in wild-type cells; translation of exogenous natural mRNA in postpolysomal extracts was somewhat lower than in wild-type preparations, but all of the other reactions and components measured were comparable to those in wild-type preparations. Preincubation of temperature-sensitive spheroplasts at 40 degrees C resulted in: a further decrease in the level of 40 S subunits; disaggregation of polysomes; loss of ability to translate natural mRNA but not poly(U); decreased ability to form 40 S preinitiation intermediates; and production of an activity, found in the cytosol, that inhibited Met-tRNA synthetase reversibly. The inhibitor had the characteristics of a protein and did not appear to be a proteinase, nuclease, or nucleotidase.     

Inhibition of peptide chain initiation by a nonhemin-regulated translational repressor from Friend leukemia cells.

A nonhemin-regulated translational repressor protein has been purified partially from the postribosomal supernatant fraction of Friend leukemia cells grown in the absence of dimethylsulfoxide. This repressor inhibits protein synthesis in lysates from rabbit reticulocytes or Friend leukemia cells and in a fractionated system using Artemia salina ribosomes, reticulocyte mRNA, and soluble components from reticulocytes. In contrast, the hemin-controlled repressor from reticulocytes does not inhibit protein synthesis in lysates from Friend leukemia cells. The repressor from Friend leukemia cells has no effect on poly(U)-directed synthesis of polyphenylalanine using reticulocyte ribosomes nor on the extension and release of nascent globin chains that were initiated in intact reticulocytes. It does not block completion of peptides on ribosomes isolated from reticulocytes incubated with NaF nor does it inhibit initiation factor-dependent formation of methionylpuromycin, but it inhibits globin mRNA-dependent methionylvaline synthesis. The Friend leukemia cell repressor promotes peptide synthesis-dependent breakdown of polysomes in reticulocyte lysates that appears to involve inhibition of ribosome reattachment to mRNA during peptide chain initiation. It is concluded that the Friend leukemia cell repressor blocks peptide initiation at a point between the addition of methionyl-tRNA^fMet to the ribosomal initiation complex and the NaF-sensitive reaction.     

Studies on the mechanism for entry of vesicular stomatitis virus glycoprotein g mRNA into membrane-bound polyribosome complexes

Glycoprotein mRNA (G mRNA) of vesicular stomatitis virus is synthesized in the cytosol fraction of infected HeLa cells. Shortly after synthesis, this mRNA associates with 40S ribosomal subunits and subsequently forms 80S monosomes in the cytosol fraction. The bulk of labeled G mRNA is then found in polysomes associated with the membrane, without first appearing in the subunit or monomer pool of the membrane-bound fraction. Inhibition of the initiation of protein synthesis by pactamycin or muconomycin A blocks entry of newly synthesized G m RNA into membrane-bound polysomes. Under these circumstances, labeled G mRNA accumulates into the cytosol. Inhibition of the elongation of protein synthesis by cucloheximide, however, allows entry of 60 percent of newly synthesized G mRNA into membrane-bound polysomes. Furthermore, prelabeled G mRNA associated with membrane-bound polysomes is released from the membrane fraction in vivo by pactamycin or mucomycon A and in vitro by 1mM puromycin - 0.5 M KCI. This release is not due to nonspecific effects of the drugs. These results demonstrate that association of G mRNA with membrane-bound polysomes is dependent upon polysome formation and initiation of protein synthesis. Therefore, direct association of the 3' end of G mRNA with the membrane does not appear to be the initial event in the formation of membrane-bound polysomes.     

Protein synthesis in oocytes of Xenopus laevis is not regulated by the supply of messenger RNA

The control of protein synthesis in oocytes of Xenopus laevis has been investigated by injecting oocytes with mRNA and polysomes followed by labeling with ¹⁴C-amino acid mixtures. Contrary to previous reports in which injected oocytes were labeled with ³H-histidine, injected globin mRNA is found to decrease amino acid incorporation into endogenous proteins competitively at all concentrations tested. No increase in overall amino acid incorporation is detected when more mRNA is supplied. Similar results are obtained after labeling injected oocytes with leucine, methionine, proline or valine individually. An explanation is presented for the conflicting results obtained when histidine is used as a label.When reticulocyte polysomes are injected, rather than purified globin mRNA, incorporation of amino acids into endogenous proteins remains roughly constant and overall incorporation increases. Similarly, when encephalomyocarditis viral RNA is injected together with either globin mRNA or reticulocyte polysomes, the globin mRNA causes decreased amino acid incorporation into encephalomyocarditis proteins, but the polysomes do not do so. The results demonstrate that different types of mRNA compete for a strictly limited translational capacity which is saturated in the normal oocyte. The limiting component is present in polysomes and is not message-specific. The constraint on protein synthesis in the amphibian oocyte cannot be fully explained by masked mRNA.     

Translational initiation regulators are hypophosphorylated in rat liver during ethionine-mediated ATP depletion

Administration of ethionine to female rats is known to inhibit hepatic protein synthesis by reducing the level of hepatic ATP. Administration of methionine and/or adenine rapidly restores the ATP levels and protein synthesis. The ethionine administration causes a progressive disaggregation of hepatic polysomes, suggesting that the initiation step of protein synthesis is inhibited. Recent studies indicate that changes in initiation are associated with alterations in the phosphorylation states of translational initiation regulators such as eukaryotic initiation factor (eIF) 4E, eIF4E-binding protein 1 (4E-BP1), and the 70-kDa ribosomal protein S6 kinase (S6K1). We found that these initiation regulators are hypophosphorylated in rat liver during ethionine-mediated ATP depletion (60% of the control value). Furthermore, the restoration of the ATP levels by the administration of methionine and adenine brought about a complete recovery of the phosphorylation states of all these regulators. The present data suggest that hypophosphorylation of various initiation regulators represents the primary event in the ethionine-induced breakdown of polysomes and inhibition of protein synthesis in the liver. Possible involvement of mammalian target of rapamycin (mTOR), as a sensor of intracellular ATP level, was also discussed.     

The binding of Met-tRNAf to isolated 40-S ribosomal subunits and the formation of Met-tRNAf - 80-S-ribosome initiation complexes.

Different forms of 40-S ribosomal subunit, distinguishable by their buoyant densities on CsCl equilibrium density gradients, are formed when derived 40-S ribosomal subunits are incubated with partially purified reticulocyte ribosomal wash proteins. One of these subunits, the 1.37-g-cm-3 form is not present in the cell but the other two forms, the 1.40-g-cm-3 and 1.40-g-cm-3 subunits, are present in cell extracts. 35S label is bound to 1.37-g-cm-3 and 1.40-g-cm-s subunits when [35S]Met-tRANf, GTP and poly(A,U,G) are included in the incubations. The 35S-labelled 40-S subunits recovered, and the amount of 35S label bound to them, are changed if the [35S]Met-tRNAf-40-S-subunit-poly(A,U,G) complexes are first purified on sucrose gradients before analysing them on CsCl. The 1.37-g-cm-3 particle is no longer seen and the total quantity of 35S label on the 40-S subunits is 90% lower after sucrose gradient purification. Between 30% and 40% of the 40-S subunits bind [35S]Met-tRNAf when 1 mM GTP, an excess of ribosomal wash proteins and [35S]Met-tRNAf over derived 40-S subunits, and poly(A,U,G) or AUG is included in the incubations. The omission of poly(A,U,G) or AUG from the incubations substantially lowers the amount of subunit-bound 35S label ultimately recovered. With these incubations less than 10% of the 40-S subunits have bound [35S]Met-tRNAf. [35S]Met-tRNAf binding is affected by the nature of the RNA added. The addition of poly(U), rRNA and native 9-S golbin mRNA is without effect, whereas denatured globin mRNA is stimulatory. Maximum binding is obtained however with AUG. Poly(A,U,G) is less stimulatory than AUG but more stimulatory than denatured mRNA, suggesting that the number as well the accessibility of the AUG initiations condons determines the amount of 35S label bound. Similar results are obtained for the ribosomal-wash-dependent binding of [35S]Met-tRNAf to 80-S ribosomes. Contrary to the binding results, the ability of mRNA to stimulate protein synthesis is dependent on the integrity of the mRNA. Thus, native 9-S globin mRNA but not poly(A,U,G) stimulatex protein synthesis in the wheat germ system. HCHO-treated globin mRNA, although stimulatory, is 45% less effective than native mRNA. The addition of AUG, derived 60-S subunits and extra ribosomal wash is required for the formation of [35S]Met-tRNAf-80-S-ribosome complexes from sucrose-gradient-purified [35S]Met-tRNAf-40-S-subunit complexes. The 80-S ribosome complexes are able to form peptide bonds. Thus, if puromycin is added to the full incubations at zero time, no 35S label is present on the 80-S ribosome. 35S label is released as methionyl-puromycin. If the [35S]Met-tRNAf-40-S-subunit complexes are assembled with poly(A,U,G) or AUG in the incubations and then purified, only derived 60-S subunits are required to form [35S]Met-tRNAf-80-S-ribosome complexes. 35S label is not released from them when puromycin is added to the incubations unless extra ribosomal wash is also added.