A translation-like cycle is a quality control checkpoint for maturing 40S ribosome subunits

Assembly factors (AFs) prevent premature translation initiation on small (40S) ribosomal subunit assembly intermediates by blocking ligand binding. However, it is unclear how AFs are displaced from maturing 40S ribosomes, if or how maturing subunits are assessed for fidelity, and what prevents premature translation initiation once AFs dissociate. Here we show that maturation involves a translation-like cycle whereby the translation factor eIF5B, a GTPase, promotes joining of large (60S) subunits with pre-40S subunits to give 80S-like complexes, which are subsequently disassembled by the termination factor Rli1, an ATPase. The AFs Tsr1 and Rio2 block the mRNA channel and initiator tRNA binding site, and therefore 80S-like ribosomes lack mRNA or initiator tRNA. After Tsr1 and Rio2 dissociate from 80S-like complexes Rli1-directed displacement of 60S subunits allows for translation initiation. This cycle thus provides a functional test of 60S subunit binding and the GTPase site before ribosomes enter the translating pool.     

The Saccharomyces cerevisiae Homologue of Mammalian Translation Initiation Factor 6 Does Not Function as a Translation Initiation Factor

Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. The Saccharomyces cerevisiae gene that encodes the 245-amino-acid eIF6 (calculated M_r 25,550), designated TIF6, has been cloned and expressed in Escherichia coli. The purified recombinant protein prevents association between 40S and 60S ribosomal subunits to form 80S ribosomes. TIF6 is a single-copy gene that maps on chromosome XVI and is essential for cell growth. eIF6 expressed in yeast cells associates with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Depletion of eIF6 from yeast cells resulted in a decrease in the rate of protein synthesis, accumulation of half-mer polyribosomes, reduced levels of 60S ribosomal subunits resulting in the stoichiometric imbalance in the 40S/60S subunit ratio, and ultimately cessation of cell growth. Furthermore, lysates of yeast cells depleted of eIF6 remained active in translation of mRNAs in vitro. These results indicate that eIF6 does not act as a true translation initiation factor. Rather, the protein may be involved in the biogenesis and/or stability of 60S ribosomal subunits.     

Occurrence of 40 S·polysomal complexes in polysome profiles of reticulocyte lysates

In most reticulocyte lysates 40 S · polysomal complexes have such a short lifetime that they will not show up in the polysome profile. Here we describe a reticulocyte lysate where these 40 S · polysomal complexes apparently have a highly increased lifetime and therefore these complexes can be seen in the polysome profile as shoulders on the di-, tri- and tetrasome peak. The presence of these complexes in lysates probably signals an increased speed in the association of 40 S subunits with mRNA since similar alterations as described above show up in the polysome profile of normal lysates to which native ribosomal 40 S subunits were added.     

Position of eukaryotic initiation factor eIF5B on the 80S ribosome mapped by directed hydroxyl radical probing

Eukaryotic translation initiation factor eIF5B is a ribosome-dependent GTPase that mediates displacement of initiation factors from the 40S ribosomal subunit in 48S initiation complexes and joining of 40S and 60S subunits. Here, we determined eIF5B's position on 80S ribosomes by directed hydroxyl radical cleavage. In the resulting model, eIF5B is located in the intersubunit cleft of the 80S ribosome: domain 1 is positioned near the GTPase activating center of the 60S subunit, domain 2 interacts with the 40S subunit (helices 3, 5 and the base of helix 15 of 18S rRNA and ribosomal protein (rp) rpS23), domain 3 is sandwiched between subunits and directly contacts several ribosomal elements including Helix 95 of 28S rRNA and helix 44 of 18S rRNA, domain 4 is near the peptidyl-transferase center and its helical subdomain contacts rpL10E. The cleavage data also indicate that binding of eIF5B might induce conformational changes in both subunits, with ribosomal segments wrapping around the factor. Some of these changes could also occur upon binding of other translational GTPases, and may contribute to factor recognition.     

Membrane-bound ribosomes of myeloma cells. II. Kinetic studies on the entry of newly made ribosomal subunits into the free and the membrane- bound ribosomal particles

The kinetics of appearance of newly made 60S and 40S ribosomal subunits in the free and membrane-bound ribosomal particles of P3K cells were explored by determining the specific radioactivities of their 18S and 28S RNA after various lengths of [3H]uridine pulse. Both 40S and 60S subunits enter free and membrane-bound polyribosomes at comparable rates from the cytoplasmic pool of newly made, free native subunits, the 40S subunits entering the native subunit pool and the polyribosomes slightly earlier than the 60S subunits. At all times, the specific radioactivity of the membrane-bound native 60S subunits was slightly lower than that of the polyribosomal 60S subunits. This indicates that the membrane-bound native 60S subunits are not precursors destined to enter membrane-bound polyribosomes and suggests that they result from the dissociation of ribosomes after chain termination. The results observed also suggest that the membrane-bound native 60S subunits are not reutilized before their release from the membranes, which probably takes place shortly after dissociation from their 40S subunits. The monoribosomes, both free and membrane-bound, had the lowest specific radioactivities in their subunits. Finally, a small amount of newly made native 40S subunits, containing 18S RNA of high specific radioactivity, and apparently also newly made messenger RNA were detected on the membranes. The high turnover of these membrane-bound native 40S subunits suggests that they may represent initiation complexes formed with mRNA which has just reached the membranes and which has not yet given rise to polyribosomes.     

Monosome Formation during Translation Initiation Requires the Serine/Arginine-Rich Protein Npl3

The yeast shuttling serine/arginine-rich protein Npl3 is required for the export of mRNAs and pre-60S ribosomal subunits from the nucleus to the cytoplasm. Here, we report a novel function of Npl3 in translation initiation. A mutation in its C terminus that prevents its dimerization (npl3Δ100) is lethal to cells and leads to translational defects, as shown by [³⁵S]methionine incorporation assays and a hypersensitivity to the translational inhibitor cycloheximide. Moreover, this Npl3 mutant shows halfmers in polysomal profiles that are indicative of defects in monosome formation. Strikingly, the loss of the ability of Npl3 to dimerize does not affect mRNA and pre-60S export. In fact, the mRNA and rRNA binding capacities of npl3Δ100 and wild-type Npl3 are similar. Intriguingly, overexpression of the dimerization domain of Npl3 disturbs dimer formation and results in a dominant-negative effect, reflected in growth defects and a halfmer formation phenotype. In addition, we found specific genetic interactions with the ribosomal subunit joining factors Rpl10 and eukaryotic translation initiation factor 5B/Fun12 and detected a substantially decreased binding of npl3Δ100 to the Rpl10-containing complex. These findings indicate an essential novel function for Npl3 in the cytoplasm, which supports monosome formation for translation initiation.     

Structure of a novel flavin chromophore from Avena coleoptiles, the possible ''blue light'' photoreceptor

The effect of edeine on the translation of mRNA or poly(U)-directed polyphenylalanine synthesis has been studied in an edeine-resistant mutant of Saccharomyces cerevisiae under three different experimental conditions: in the whole lysate system, in a micrococcal-nuclease-treated lysate, and in a high-salt-treated lysate. The results indicate that translation of messenger is more resistant to edeine in the whole lysate than in the depleted lysates; these observations suggest that resistance to edeine is associated with the presence of endogenous mRNA. It is shown that 40S mutant subunits have a higher affinity for polysomal RNA than 40S wild-type subunits. Since the mRNA binding is inhibited by 7-methylguanosine 5'-monophosphate, the interaction between polysomal RNA and 40S ribosomes is specific for mRNA. The data demonstrate that in each of the depleted lysates, with edeine initially present, the formation of the 80S initiation complex is inhibited. However, edeine inhibition of [3H]methionine binding to 80S ribosomes is overcome completely in the mutant extract by preincubation of this lysate with polysomal RNA. The results indicate that the mutant may carry a specific change in a messenger-binding factor or in a ribosomal protein thereby permitting an increased stability of the messenger-ribosome complex which consequently results in an increased resistance of the mutant lysate to edeine.     

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.     

Assembly of 60S ribosomal subunits is perturbed in temperature-sensitive yeast mutants defective in ribosomal protein L16.

Temperature-sensitive mutants defective in 60S ribosomal subunit protein L16 of Saccharomyces cerevisiae were isolated through hydroxylamine mutagenesis of the RPL16B gene and plasmid shuffling. Two heat-sensitive and two cold-sensitive isolates were characterized. The growth of the four mutants is inhibited at their restrictive temperatures. However, many of the cells remain viable if returned to their permissive temperatures. All of the mutants are deficient in 60S ribosomal subunits and therefore accumulate translational preinitiation complexes. Three of the mutants exhibit a shortage of mature 25S rRNA, and one accumulates rRNA precursors. The accumulation of rRNA precursors suggests that ribosome assembly may be slowed in this mutant. These phenotypes lead us to propose that mutants containing the rpl16b alleles are defective for 60S subunit assembly rather than function. In the mutant carrying the rpl16b-1 allele, ribosomes initiate translation at the noncanonical codon AUA, at least on the rpl16b-1 mRNA, bringing to light a possible connection between the rate and the fidelity of translation initiation.     

Feedback inhibition of poly(A)-binding protein mRNA translation. A possible mechanism of translation arrest by stalled 40 S ribosomal subunits

An adenine-rich cis element at the 5'-untranslated region (UTR) of Pabp1 mRNA is able to inhibit translation of its own mRNA. Similar inhibition of translation of a reporter beta-galactosidase mRNA is observed when the adenine-rich auto regulatory sequence (ARS) is placed within the 5'-UTR of this mRNA. For this translational control the distance of the ARS from the 5' cap is not important. However, it determines the number of 40 S ribosomal subunits bound to the translationally arrested mRNA. Inhibition of mRNA translation by this regulatory sequence occurs at the step of joining of the 60 S ribosomal subunit to the pre-initiation complex. Translational arrest of the ARS containing mRNA in a rabbit reticulocyte lysate cell-free system in the presence of exogenous Pabp1 protects the 5'-flanking region of the ARS from nuclease digestion. This protection depends on the binding of the 40 S ribosomal subunit to the mRNA. The size and the sequence of the nucleotide-protected fragment depends on the location of the ARS within the 5'-UTR. When the ARS is located at a distance of about 78 nucleotides from the 5' cap, a 40-nucleotide long region adjacent to the ARS is protected. On the other hand, when the ARS is moved further away from the 5' cap to a distance of approximately 267 nucleotides, a 100-nucleotide-long region adjacent to the ARS is protected from nuclease digestion. Nuclease protection is attributed to the presence of one or more stalled 40 S ribosomal subunits near the Pabp1-bound ARS.     

Importance of polysomal mRNA-associated polypeptides for protein synthesis initiation in yeast

The polysomal mRNA from the cell-free system of the yeast Saccharomyces cerevisiae, in the absence of exogenous energy, binds to the 40S ribosomal subunit thus forming a 48S preinitiation complex which, with energy added, is converted into 80S initiation complex. By using ribosomes with a high affinity to polysomal mRNA (pmRNA) from an edeine-resistant mutant of S. cerevisiae in place of wild-type ribosomes, increased quantities of the 48S preinitiation complex are obtained. The pmRNA is found associated with several polypeptides having molecular masses of 115-98 kDa, 72 kDa, 60 kDa and 51 kDa. These polypeptides, labelled with 125I, interact with 40S and 80S ribosomes and are essential for the formation of the 48S and 80S initiation complexes inasmuch as deproteinized pmRNA alone cannot initiate the process. In addition, other polypeptides present in the cytosol are required to carry out the above-mentioned steps of protein synthesis.     

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.     

Inhibition by suramin of protein synthesis in vitro. Ribosomes as the target of the drug

Suramin, a drug widely used both as a therapeutic agent and in research, inhibits translation in eukaryotic cell-free systems from rabbit reticulocyte lysate (IC(50)=142-241 microM). Suramin affects both initiation (block of 43S pre-initiation complex formation) and elongation (impairment of poly(U) translation). The drug induces an increase in the pools of ribosomal subunits and the formation of high molecular weight ribosomal complexes, thus causing the disappearance of polysomes. Ribosomes isolated from suramin-treated translating mixtures are inactivated. [(3)H]Suramin binds to ribosomes and to isolated 60S and 40S ribosomal subunits (116, 106 and 3 binding sites, respectively) showing higher affinity for the small subunit (K(d)=2 microM).     

Yeast virus propagation depends critically on free 60S ribosomal subunit concentration.

Over 30 MAK (maintenance of killer) genes are necessary for propagation of the killer toxin-encoding M1 satellite double-stranded RNA of the L-A virus. Sequence analysis revealed that MAK7 is RPL4A, one of the two genes encoding ribosomal protein L4 of the 60S subunit. We further found that mutants with mutations in 18 MAK genes (including mak1 [top1], mak7 [rpl4A], mak8 [rpl3], mak11, and mak16) had decreased free 60S subunits. Mutants with another three mak mutations had half-mer polysomes, indicative of poor association of 60S and 40S subunits. The rest of the mak mutants, including the mak3 (N-acetyltransferase) mutant, showed a normal profile. The free 60S subunits, L-A copy number, and the amount of L-A coat protein in the mak1, mak7, mak11, and mak16 mutants were raised to the normal level by the respective normal single-copy gene. Our data suggest that most mak mutations affect M1 propagation by their effects on the supply of proteins from the L-A virus and that the translation of the non-poly(A) L-A mRNA depends critically on the amount of free 60S ribosomal subunits, probably because 60S association with the 40S subunit waiting at the initiator AUG is facilitated by the 3' poly(A).     

Effect of Met-tRNAf deacylase on polypeptide chain initiation in rabbit reticulocyte lysate

The possible role of Met-tRNAf deacylase in the regulation of protein synthesis in rabbit reticulocyte lysate by the hemin-controlled translational repressor (HCR) or the double-stranded RNA-activated inhibitor (dsI) has been examined. Inhibition of protein synthesis by either HCR or dsI is associated with a marked increase in the steady state level of 48 S initiation complexes, containing a 40 S ribosomal subunit, globin mRNA, and a reduced level of Met-tRNAf, suggesting that the rate of 60 S subunit addition may be inhibited and that subunit-bound Met-tRNAf may become deacylated by Met-tRNAf deacylase. The addition of highly purified Met-tRNAf deacylase to lysate samples incubated with HCR or dsI reduces the [35S]Met-tRNAf labeling of 48 S complexes to even a lower level but has no effect on the high level of [35S]Met-tRNAf associated with 43 S complexes in the plus hemin control. The effect of added deacylase on the labeling of 48 S complexes with [35S]Met-tRNAf can be overcome by adding eIF-5 or a soluble reticulocyte protein that has been termed the reversing factor, but not by the addition of eIF-2. Added deacylase has no effect on the level of mRNA in 48 S complexes or the labeling of these complexes with [35S]fMet-tRNAf. When lysate samples were labeled with Met-tRNAf, purified from wheat germ or yeast, and doubly labeled with 32P at the 5' end and [35S]methionine aminoacylation, HCR reduced the level of 32P and 35S-labeled tRNAMetf in 48 S complexes to a similar degree, suggesting that once it has become deacylated, tRNAMetf dissociates from the 40 S subunit.     

Effects of eucaryotic initiation factor 3 on eucaryotic ribosomal subunit equilibrium and kinetics

In order to understand the possible role of eucaryotic initiator factor 3 (eIF-3) in maintaining a pool of eucaryotic subunits, we have measured the effects of eIF-3 on the equilibria and kinetics of ribosomal subunit association and dissociation. The ribosomal subunit interactions have been studied by laser light scattering, which does not perturb the system. We find that eIF-3 reduces the apparent association rate of reticulocyte, wheat germ, and Artemia ribosomes. The kinetics of the reassociation for a shift in [Mg2+] from 0.5 to 6 mM are best explained by a model where eIF-3 dissociates from the 40S subunits prior to association of the 40S and 60S subunits. Static titrations indicate there is some binding of eIF-3 to 80S ribosomes at lower [Mg2+].     

Translational control mechanism of ornithine decarboxylase by asparagine and putrescine in primary cultured hepatocytes

Asparagine stimulated the translation of ornithine decarboxylase (ODC) mRNA more than 10-fold in cultured hepatocytes which had been pretreated with glucagon in simple salt/glucose medium. Putrescine suppressed the increase in the rate of ODC synthesis caused by asparagine without significant change in the amount of ODC mRNA, suggesting that putrescine inhibited the effect of asparagine at least in part at the level of translation. Polysomal distribution of ODC mRNA was analyzed to examine the site of translational regulation by these effectors. In uninduced hepatocytes, most of the ODC mRNA was sedimented slightly after the 40 S ribosomal subunit. This ODC mRNA was sequestered from translational machinery since it was not shifted to the polysome fraction when peptide elongation was specifically inhibited by a low concentration of cycloheximide. In asparagine-treated cells, 40% of total ODC mRNA was in the polysomal fraction and formed heavier polysomes, indicating that asparagine stimulated both recruitment of ODC mRNA from the untranslatable pool and the initiation steps of translation. Putrescine did not change the distribution pattern of ODC mRNA on polysomes significantly. Thus, 30% of ODC mRNA remained on polysomes even when ODC synthesis was completely inhibited by putrescine. Paradoxically more than 70% of ODC mRNA was shifted into polysomes by putrescine in the presence of low concentrations of cycloheximide. These results, together with changes in the polysome profile, suggested that putrescine nonspecifically stimulated the recruitment of ODC mRNA from the untranslatable pool, whereas it specifically inhibited its translation at both the initiation and the elongation steps.     

Influence of the state of ribosome association on the phosphorylation of ribosomal proteins in isolated ribosome--protein kinase systems from rat cerebral cortex.

Ribosomal protein phosphorylation was investigated in isolated ribosomal subunits and polyribosomes from rat cerebral cortex in the presence of [gamma-32P]ATP and purified catalytic subunit of cyclic AMP-dependent protein kinase from the same tissue. Ribosomal proteins that were most readily phosphorylated in isolated cerebral ribosomal subunits included proteins S2, S3a, S6 and S10 of the 40 S subunit and proteins L6, L13, L14, L19 and L29 of the 60 S subunit. These proteins were also phosphorylated in cellular preparations of rat cerebral cortex in situ or in vitro [Roberts & Ashby (1978) J. Biol. Chem. 253, 288-296; Roberts & Morelos (1979) Biochem. J. 184, 233-244]. However, several additional ribosomal proteins were phosphorylated when isolated 40 S or 60 S subunits were separately incubated in the reconstituted system. Analogous results were obtained with an equimolar mixture of cerebral 40 S and 60 S subunits under comparable conditions. In contrast, extensive exposure of purified cerebral polyribosomes to the catalytic subunit resulted in phosphorylation of only those ribosomal proteins of the 40 S subunit that were most highly labelled after the administration of [32P]Pi in vivo: proteins S2, S6 and S10. Ribosomal proteins of 60 S subunits that were readily phosphorylated in isolated cerebral polyribosomes included proteins L6, L13 and L29. These results indicate that polyribosome formation markedly decreases the number of ribosomal protein sites available for phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase. Moreover, the findings suggest that, of the ribosomal protein phosphorylations observed in rat cerebral cortex in vivo, proteins S2, S6, S10, L6, L13 and L29 can be phosphorylated in polyribosomes, whereas proteins S3a, S5, L14 and L19 may become phosphorylated only in free ribosomal subunits.