The 60 S ribosomal subunit as a carrier of eukaryotic initiation factor 2 and the site of reversing factor activity during protein synthesis

Studies on the recycling of eukaryotic initiation factor 2 (eIF-2) during protein synthesis in normal and heme-deficient reticulocyte lysates indicate that eIF-2 binds physiologically to the 60 S ribosomal subunit. Several findings suggest that the 60 S subunit serves as a carrier for eIF-2 during protein synthesis. The addition of purified eIF-2 (beta-32P) to normal hemin-supplemented lysates results in its binding to polyribosomal 60 S subunits; the binding is temperature-dependent. In lysates inhibited by heme deficiency, phosphorylated eIF-2 alpha can be detected on polyribosomal 60 S subunits early in the initial linear phase of protein synthesis; after polyribosomal disaggregation and shut-off of protein synthesis, phosphorylated eIF-2 alpha accumulates on free 60 S ribosome subunits and on the 60 S subunits of 80 S ribosome couples. The phosphorylated eIF-2 alpha associated with the 60 S subunits in heme-deficient lysates appears to be present as the binary complex [eIF-2 (alpha P) X GDP]; the binding of this complex to the 60 S subunit is tight and is not affected by treatment with 25 mM EDTA or by sedimentation in sucrose gradients. Reversal of the inhibition of protein synthesis in heme-deficient lysates by the addition of reversing factor results in a rapid binding of reversing factor to the 60 S subunits and a concomitant dissociation of [eIF-2(alpha P) X GDP]. These findings suggest that the [eIF-2 X GDP] binary complex formed during the assembly of the 80 S initiation complex binds to the 60 S subunit of polyribosomes and is subsequently released by the action of reversing factor.     

Initiation of mammalian protein synthesis. II. The assembly of the initiation complex with purified initiation factors

The assembly of initiation complexes is studied in a protein synthesis initiation assay containing ribosomal subunits, globin [¹²⁵I]mRNA, [³H]Met-tRNA_f, seven purified initiation factors, ATP and GTP. By omitting single components from the initiation assay, specific roles of the initiation factors, ATP and GTP are demonstrated. The initiation factor eIF-2 is required for the binding of Met-tRNA_f to the 40 S ribosomal subunit. The initial Met-tRNA_f binding to the small ribosomal subunit is a stringent prerequisite for the subsequent mRNA binding. The initiation factors eIF-3, eIF-4A, eIF-4B and eIF-4C together with ATP promote the binding of mRNA to the 40 S initiation complex. The association of the 40 S initiation complex with the 60 S ribosome subunit to form an 80 S initiation complex is mediated by the initiation factor eIF-5 and requires the hydrolysis of GTP. The factor eIF-1 gives a twofold overall stimulation of initiation complex formation. A model of the sequential steps in the assembly of the 80 S initiation complex in mammalian protein synthesis is presented.     

Evidence that the primary effect of phosphorylation of eukaryotic initiation factor 2(alpha) in rabbit reticulocyte lysate is inhibition of the release of eukaryotic initiation factor-2.GDP from 60 S ribosomal subunits

The phosphorylation of eukaryotic initiation factor (eIF) 2 alpha that occurs when rabbit reticulocyte lysate is incubated in the absence of hemin or with poly(I.C) causes inhibition of polypeptide chain initiation by preventing a separate factor (termed RF) from promoting the exchange of GTP for GDP on eIF-2. When lysate was incubated in the presence of hemin and [14C] eIF-2 or [alpha-32P]GTP, we observed binding of eIF-2 and GDP or GTP to 60 S ribosomal subunits that was slightly greater than that bound to 40 S subunits and little binding to 80 S ribosomes. When incubation was in the absence of hemin or in the presence of hemin plus 0.1 microgram/ml poly(I.C), eIF-2 and GDP binding to 60 S subunits was increased 1.5- to 2-fold, that bound to 80 S ribosomes was almost as great as that bound to 60 S subunits, and that bound to 40 S subunits was unchanged. Our data indicate that about 40% of the eIF-2 that becomes bound to 60 S subunits and 80 S ribosomes in the absence of hemin or with poly(I.C) is eIF-2(alpha-P) and suggest that the eIF-2 and GDP bound is probably in the form of a binary complex. The accumulation of eIF-2.GDP on 60 S subunits occurs before binding of Met-tRNAf to 40 S subunits becomes reduced and before protein synthesis becomes inhibited. The rate of turnover of GDP (presumably eIF-2.GDP) on 60 S subunits and 80 S ribosomes in the absence of hemin is reduced to less than 10% the control rate, because the dissociation of eIF-2.GDP is inhibited. Additional RF increases the turnover of eIF-2.GDP on 60 S subunits and 80 S ribosomes to near the control rate by promoting dissociation of eIF-2.GDP but not eIF-2(alpha-P).GDP. Our findings suggest that eIF-2.GTP binding to and eIF-2.GDP release from 60 S subunits may normally occur and serve to promote subunit joining. The phosphorylation of eIF-2 alpha inhibits polypeptide chain initiation by preventing dissociation of eIF-2.GDP from either free 60 S subunits (thus inhibiting subunit joining directly) or the 60 S subunit component of an 80 S initiation complex (thereby blocking elongation and resulting in the dissociation of the 80 S complex).     

A rapid and sensitive assay for the detection of eukaryotic ribosome dissociation factors.

A rapid and sensitive assay has been developed for the factor-dependent dissociation of eukaryotic ribosomes. This assay takes advantage of the observation that initiation factor eIF-2 will bind Met-tRNA_f^met to 40 S subunits but not to 80 S ribosomes. Incubation of wheat germ ribosomes at 1 mm Mg²⁺ results in their dissociation into 40 S subunits. These subunits spontaneously reassociate when the Mg²⁺ concentration is raised to 4 mm. However, if the incubation at 1 mm Mg²⁺ is carried out in the presence of an extract containing a ribosome dissociation factor, a certain portion of the subunits will fail to reassociate when the Mg²⁺ concentration is raised to 4 mm. The 40 S subunits remaining due to the presence of the dissociation factor can bind [³⁵S]Met-tRNA_f^met in the presence of wheat germ eIF-2. The [³⁵S]Met-tRNA_f^met bound to the 40 S subunits is readily detected by its retention on a Millipore filter.     

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+].     

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.     

Purification and characterization of a ribosome dissociation factor (eukaryotic initiation factor 6) from wheat germ.

A wheat germ ribosome dissociation factor, eukaryotic initiation factor 6 (eIF-6), has been purified almost to homogeneity from the 25 to 40% ammonium sulfate fraction of the postribosomal supernatant. This dissociation factor is distinct from initiation factor eIF-3 and its chromatographic properties permit its separation from the known wheat germ initiation factors. Under certain conditions, eIF-6 stimulates the incorporation of amino acids into polypeptides in a partially fractionated wheat germ cell-free system. The eight-step purification procedure developed includes chromatography on DEAE-cellulose, phosphocellulose, Sephadex G-75, and hydroxyapatite and yields a dissociation factor more than 80% pure. The purified factor is composed of a single polypeptide chain with a molecular weight of approximately 23,000 as determined by gel filtration chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is an acidic protein which is heat labile and is inactivated by treatment with N-ethylmaleimide. The dissociation factor is much more effective in preventing the reassociation of 40 S and 60 S ribosomal subunits than in directly dissociating 80 S ribosomes. Like Escherichia coli IF-3, about 10 pmol of the dissociation factor are required to dissociate 1 pmol of ribosomes.     

Role of eukaryotic initiation factor 5 in the formation of 80 S initiation complexes.

The function of eukaryotic initiation factor 5 (eIF-5) from rabbit reticulocyte lysate has been studied by sucrose gradient preparation of 40 S and 80 S initiation complexes. eIF-5 is required for transfer of initiator tRNA from 40 S preinitiation complexes to puromycin-reactive 80 S complexes. The transfer is dependent upon GTP hydrolysis and is associated with release of eIF-2 and eIF-3 from the 40 S subunit. The GTP-dependent loss of eIF-2 and eIF-3 is catalyzed by eIF-5 in the absence of 60 S subunits or when subunit joining is prevented by edeine, but not when GTP is replaced by GuoPP(NH)P. Unstable 40 S subunit . Met-tRNAf complexes generated by eIF-5 can form puromycin-reactive 80 S complexes when 60 S subunits are added in the absence of added GTP. In addition, kinetic evidence is presented that indicates GTP hydrolysis occurs prior to 80 S complex formation.     

Studies on native ribosomal subunits from rat liver. Evidence for activities associated with native 40S subunits that affect the interaction with acetylphenylalanyl-tRNA, methionyl-tRNAf, and 60S subunits.

The binding of the initiator tRNA Met-tRNAf, and of acetylphenylalanyl-tRNA, has been examined with rat liver 40S subunits derived from 80S ribosomes by dissociation with native 40S subunits sedimented from the postmicrosomal fraction and with native 40S subunits extracted with high salt-containing solutions. Binding of Met-tRNAf and acetylphenylalanyl-tRNA to derived and to salt-extracted native 40S subunits is observed in the presence of the appropriate polynucleotide template and a highly purified binding factor obtain from the soluble fraction of rat liver homogenates (R.L. IF-1). Native 40S subunits bind acetylphenylalanyl-tRNA in a reaction that requires poly(U) but not exogenous binding factor; however, Met-tRNAf is not bound to native subunits, even when supplemented with the soluble binding factor, or under conditions where factor-independent, high Mg2+-stimulated binding is observed with the derived and the salt-washed native 40S subunits. The extract obtained from native 40S subunits promotes the binding of acetylphenylalanyl-tRNA but not Met-tRNAf to derived and to salt-extracted native subunits. The addition of native 40S extract to incubations containing R.L. IF-1, Met-tRNAf, and derived 40S subunits, inhibits the formation of 40S-Met-tRNAf complex. These data suggest that the binding activity that is specific for 40S subunits and initiator tRNA, and an activity that inhibits the interaction with Met-tRNAf specifically, are both associated with native 40S subunits, and can be extracted from them by treatment with high salt-containing solutions. Derived 40S subunits react quantitatively with 60S particles to form 80S ribosomes which do not bind acetylphenylalanyl-tRNA with binding factor R.L. IF-1. Native 40S subunits react only partly with 60S subunits; about half of the native 40S subunit population forms 80S ribosomes which do not subsequently bind acetylphenylalanyl-tRNA; the remaining native 40S subunits which do not react with 60S particles bind acetylphenylalanyl-tRNA but to a lesser extent. When preformed native 40S-acetylphenylalanyl-tRNA complex is incubated with 60S subunits, about half of the subunits form an 80S-acetylphenylalanyl-tRNA complex, while the rest remains as 40S-acetylphenylalanyl-tRNA. The addition of native 40S subunit salt extract to incubations containing preformed 80S ribosomes dissociates the particles to subunits. These data suggest that in addition to the initiator tRNA binding activity and the activity that inhibits Met-tRNAf interaction, part of the native 40S subunit population also contains an activity that dissociates 80S ribosomes.     

Binding and release of radiolabeled eukaryotic initiation factors 2 and 3 during 80 S initiation complex formation.

The AUG-dependent formation of an 80 S ribosomal initiation complex was studied using purified rabbit reticulocyte initiation factors radiolabeled by reductive methylation. The radiolabeled initiation factors were as biologically active as untreated factors. Reaction mixtures containing a variety of components (AUG, GTP, Met-tRNAf, initiation factors, and 40 S and 60 S ribosomal subunits) were incubated at 30 degrees C and then analyzed on linear sucrose gradients for the formation of ribosomal complexes. The results show that both eukaryotic initiation factor (eIF)-3 and the ternary complex (eIF-2.GTP.Met-tRNAf) bind independently to the 40 S subunit and each of these components enhances the binding of the other. All of the polypeptides of eIF-2 and eIF-3 participate in this binding. Formation of an 80 S ribosomal complex requires eIF-5 and 60 S subunits in a reaction that is stimulated by eIF-4C. Both eIF-2 and eIF-3 are released from the 40 S preinitiation complex during formation of the 80 S initiation complex. Release of eIF-2 and eIF-3 does not occur and 80 S ribosomal complexes are not formed if GTP is replaced by a nonhydrolyzable analog such as guanosine 5'-O3-(1,2-mu-imido)triphosphate. Despite a variety of attempts, it has not yet been possible to demonstrate binding of eIF-4C, eIF-4D, or eIF-5 to either 40 S or 80 S ribosomal complexes.     

Release and recycling of eukaryotic initiation factor 2 in the formation of an 80 S ribosomal polypeptide chain initiation complex.

The eukaryotic initiation factor (eIF)-5 mediates hydrolysis of GTP bound to the 40 S initiation complex in the absence of 60 S ribosomal subunits. The eIF-2.GDP formed under these conditions is released from the 40 S ribosomal subunit while initiator Met-tRNA(f) remains bound. The released eIF-2.GDP can participate in an eIF-2B-catalyzed GDP/GTP exchange reaction to reform the Met-tRNA(f).eIF-2.GTP ternary complex. In contrast, when 60 S ribosomal subunits were also present in an eIF-5-catalyzed reaction, the eIF-2.GDP produced remained bound to the 60 S ribosomal subunit of the 80 S initiation complex. When such an 80 S initiation complex, containing bound eIF-2.GDP, was incubated with GTP and eIF-2B, GDP was released. However, eIF-2 still remained bound to the ribosomes and was unable to form a Met-tRNA(f)l.eIF-2.GTP ternary complex. In contrast, when 60 S ribosomal subunits were preincubated with either free eIF-2 or with eIF-2.eIF-2B complex and then added to a reaction containing both the 40 S initiation complex and eIF-5, the eIF-2.GDP produced did not bind to the 60 S ribosomal subunits but was released from the ribosomes. Thus, the 80 S initiation complex formed under these conditions did not contain bound eIF-2.GDP. Under similar experimental conditions, preincubation of 60 S ribosomal subunits with purified eIF-2B (free of eIF-2) failed to cause release of eIF-2.GDP from the ribosomal initiation complex. These results suggest that 60 S ribosome-bound eIF-2.GDP does not act as a direct substrate for eIF-2B-mediated release of eIF-2 from ribosomes. Rather, the affinity of 60 S ribosomal subunits for either eIF-2, or the eIF-2 moiety of the eIF-2.eIF-2B complex, prevents association of 60 S ribosomal subunits with eIF-2.GDP formed in the initiation reaction. This ensures release of eIF-2 from ribosomes following hydrolysis of GTP bound to the 40 S initiation complex.     

Function of eukaryotic initiation factor 5 in the formation of an 80 S ribosomal polypeptide chain initiation complex.

Eukaryotic initiation factor 5 (eIF-5), isolated from rabbit reticulocyte lysates, is a monomeric protein of 58-62 kDa. The function of eIF-5 in the formation of an 80 S polypeptide chain initiation complex from a 40 S initiation complex has been investigated. Incubation of the isolated 40 S initiation complex (40 S.AUG.Met.tRNAf.eIF-2 GTP) with eIF-5 resulted in the rapid and quantitative hydrolysis of GTP bound to the 40 S initiation complex. The rate of this reaction was unaffected by the presence of 60 S ribosomal subunits. Analysis of eIF-5-catalyzed reaction products by gel filtration indicated that both eIF-2.GDP binary complex and Pi formed were released from the ribosomal complex whereas Met-tRNAf remained bound to 40 S ribosomes as a Met-tRNAf.40 S.AUG complex. Reactions carried out with biologically active 32P-labeled eIF-5 indicated that this protein was not associated with the 40 S.AUG.Met-tRNAf complex; similar results were obtained by immunological methods using monospecific anti-eIF-5 antibodies. The isolated 40 S.AUG.Met-RNAf complex, free of eIF-2.GDP binary complex and eIF-5, readily interacted with 60 S ribosomal subunits in the absence of exogenously added eIF-5 to form the 80 S initiation complex capable of transferring Met-tRNAf into peptide linkages. These results indicate that the sole function of eIF-5 in the initiation of protein synthesis is to mediate hydrolysis of GTP bound to the 40 S initiation complex in the absence of 60 S ribosomal subunits. This leads to formation of the intermediate 40 S.AUG.Met-tRNAf and dissociation of the eIF-2.GDP binary complex. Subsequent joining of 60 S ribosomal subunits to the intermediate 40 S.AUG.Met-tRNAf complex does not require participation of eIF-5. Thus, the formation of an 80 S ribosomal polypeptide chain initiation complex from a 40 S ribosomal initiation complex can be summarized by the following sequence of partial reactions. (40 S.AUG.Met-tRNAf.eIF-2.GTP) eIF-5----(40 S.AUG.Met-tRNAf) + (eIF-2.GDP) + Pi (1) (40 S.AUG.Met-tRNAf) + 60 S----(80 S.AUG.Met-tRNAf) (2) 80 S initiation complex.     

Structural Insights into ribosome recycling factor interactions with the 70S ribosome

At the end of translation in bacteria, ribosome recycling factor (RRF) is used together with elongation factor G to recycle the 30S and 50S ribosomal subunits for the next round of translation. In x-ray crystal structures of RRF with the Escherichia coli 70S ribosome, RRF binds to the large ribosomal subunit in the cleft that contains the peptidyl transferase center. Upon binding of either E. coli or Thermus thermophilus RRF to the E. coli ribosome, the tip of ribosomal RNA helix 69 in the large subunit moves away from the small subunit toward RRF by 8 Å, thereby disrupting a key contact between the small and large ribosomal subunits termed bridge B2a. In the ribosome crystals, the ability of RRF to destabilize bridge B2a is influenced by crystal packing forces. Movement of helix 69 involves an ordered-to-disordered transition upon binding of RRF to the ribosome. The disruption of bridge B2a upon RRF binding to the ribosome seen in the present structures reveals one of the key roles that RRF plays in ribosome recycling, the dissociation of 70S ribosomes into subunits. The structures also reveal contacts between domain II of RRF and protein S12 in the 30S subunit that may also play a role in ribosome recycling.     

Evidence that a single GTP is used in the formation of 80 S initiation complexes.

Evidence is presented that the GTP initially bound in ternary complex (Met-tRNAf.GTP.eukaryotic initiation factor 2 (eIF-2)) is the same GTP that is hydrolyzed to allow joining of a 40 S preinitiation complex with 60 S subunits. This evidence was obtained by two quite dissimilar techniques. The first was a kinetic analysis of AUG-directed methionyl-puromycin synthesis using either eIF-2 of eIF-2A to direct the binding of Met-tRNAf to 40 S subunits. The second technique was the isolation of 40 S preinitiation complexes by Sepharose 6B chromatography and subsequent quantitation of GTP hydrolysis and methionyl-puromycin synthesis under conditions where 80 S complex formation is permitted.     

Formation and release of eukaryotic initiation factor 2 X GDP complex during eukaryotic ribosomal polypeptide chain initiation complex formation

The formation and release of an eukaryotic initiation factor (eIF)-2 X GDP binary complex during eIF-5-mediated assembly of an 80 S ribosomal polypeptide chain initiation complex have been studied by sucrose gradient centrifugation analysis. Isolated 40 S initiation complex reacts with eIF-5 and 60 S ribosomal subunits to form an 80 S ribosomal initiation complex with concomitant hydrolysis of an equimolar amount of bound GTP to GDP and Pi. Sucrose gradient analysis of reaction products revealed that GDP was released from ribosomes as an eIF-2 X GDP complex. Evidence is presented that eIF-5-mediated hydrolysis releases the GTP bound to the 40 S initiation complex as an intact eIF-2 X GDP complex rather than as free GDP and eIF-2 which subsequently recombine to form the binary complex. Furthermore, formation and release of eIF-2 X GDP from the ribosomal complex do not require concomitant formation of an 80 S initiation complex since both reactions occur efficiently when the 40 S initiation complex reacts with eIF-5 in the absence of 60 S ribosomal subunits. These results, along with the observation that the 40 S initiation complex formed with the nonhydrolyzable analogue of GTP, 5'-guanylylmethylene diphosphonate, can neither join a 60 S ribosomal subunit nor releases ribosome-bound eIF-2, suggest that following eIF-5-mediated hydrolysis of GTP bound to the 40 S initiation complex, both Pi and eIF-2 X GDP complex are released from ribosomes prior to the joining of 60 S ribosomal subunits to the 40 S initiation complex.     

Binding of spectinomycin by ribosomes fromEscherichia coli

The binding of the aminocyclitol antibiotic spectinomycin to 70S ribosomes and to 30S subunits fromEscherichia coli has been investigated. The association was influenced by the presence of messenger RNA. The K_d for [³H]-4 OH-spectinomycin binding to 70S ribosomes was 2×10^–7 M without mRNA (polyinosinic acid), and 1×10^–6 M with polyinosinic acid. Dissociation of the antibiotic from the ribosomes was significantly affected by the presence of a bound messenger RNA, which reduced the rate of dissociation by a factor of 5.7. The presence of mRNA did not influence the association of spectinomycin with the 30S subunit. The dissociation rate from the small subunit was comparable to the rate of dissociation from the 70S ribosome and was not affected by the presence of mRNA.     

Structural dynamics of bacterial ribosomes: IV. Classification of ribosomes by subunit interaction

Previous studies in this series (M. Noll et al., 1973a,b; Noll & Noll, 1974) have established that in Escherichia coli the ability of subunits to form vacant 70 S ribosome couples at 10 mm-Mg²⁺ is a stringent condition for activity in the translation of natural messenger (R17 RNA). The present study examines the structural basis of subunit interaction. It is found that vacant ribosome couples prepared by various methods fall into two classes, “tight” couples and “loose” couples, that differ in the affinity of their subunits for each other. Detection and separation of the two particle species is possible by ultracentrifugation. When analyzed on sucrose gradients at 6 mm-Mg²⁺ and moderate speed (30,000 revs/min), tight couples sediment as undissociated 70 S ribosomes, whereas loose couples are completely dissociated and sediment as 30 S and 50 S subunits. At 15 mm-Mg²⁺ in the gradient, both species sediment as a 70S peak. At 10 mm-Mg²⁺ and 60,000 revs/min, two peaks (63 S and 55 S) are seen because the high hydrostatic pressure causes more pronounced dissociation of the loose than of the tight couples.Association is dependent on the state of each subunit. Removal of Mg²⁺ produces 30 S b-particles that are unable to associate with 50 S subunits unless reconverted to the 30 S a-form by thermal activation according to Zamir et al. (1971). In the dissociated state, 50 S subunits tend to change irreversibly to a 50 S b-modification that produces loose couples upon association with 30 S a-subunits. The 50 S a → 50 S b transition could not be related to breaks in 23 S RNA detectable by sedimentation analysis. However, mild treatment of 50 S a-subunits with RNase produces particles that associate with 30 S a-subunits to couples that are less stable than the loose couples resulting from a dissociation/association step.Fresh S-30 extracts contain only tight couples (approx. 80%) and subunits (approx. 20%). Our results suggest that loose couples are artefacts derived from tight couples by a structural or conformational modification.Interaction-free subunits that previously were found to form a primitive initiation complex with poly(U) and tRNA_Phe (Schreier & Noll, 1970,1971), and to be active in phenylalanine polymerization, are shown to consist of the b-form of each subunit.It is likely that conflicting results obtained in the study of the mechanism of initiation and other aspects of ribosome function are due to the lack of structural criteria required for standardizing the ribosome preparation used by different investigators. This study provides simple methods and criteria to classify and separate physically all ribosome and ribosome subunits that have been observed into well-defined classes of predictable activity.     

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.     

Eukaryotic initiation factor 5 from calf liver is a single polypeptide chain protein of Mr = 62,000

Eukaryotic initiation factor 5 (eIF-5), which specifically catalyzes the joining of a 60 S ribosomal subunit to a 40 S initiation complex to form a functional 80 S initiation complex, has been purified from ribosomal salt wash proteins of calf liver. The purified factor exhibits only one polypeptide band of Mr = 62,000 following electrophoresis in 10% polyacrylamide gels in the presence of sodium dodecyl sulfate. The native protein has a sedimentation coefficient of 4.2 S and a Stokes radius of 33 A which is consistent with eIF-5 being a monomeric protein of Mr = 58,000-62,000. Less pure preparations of eIF-5 elute in gel filtration columns with an apparent Mr of 160,000-180,000 presumably due to association of eIF-5 with other high molecular weight proteins since eIF-5 activity present in such preparations can also be shown by gel electrophoretic separation under denaturing conditions to be associated with a 62,000-dalton protein. Furthermore, eIF-5 purified from calf liver extracts with or without a number of protease inhibitors is indistinguishable with regard to molecular weight and final specific activity of purified preparations. The purified factor catalyzes the hydrolysis of GTP present in 40 S initiation complexes in the absence of 60 S ribosomal subunits. The presence of 60 S ribosomal subunits neither stimulates nor inhibits the hydrolysis of GTP. However, the factor cannot mediate 40 S or 40 + 60 S ribosome-dependent hydrolysis of GTP in the absence of Met-tRNAf or other components required for 40 S initiation complex formation. It can be calculated that 1 pmol of eIF-5 protein can catalyze the formation of at least 10 pmol of 80 S initiation complex under the conditions of in vitro initiation reactions.     

Phosphorylation of wheat germ initiation factors and ribosomal proteins

The ability of the wheat germ initiation factors and ribosomes to serve as substrates for a wheat germ protein kinase (Yan and Tao 1982 J Biol Chem 257: 7037-7043) has been investigated. The wheat germ kinase catalyzes the phosphorylation of the 42,000 dalton subunit of eukaryotic initiation factor (eIF)-2 and the 107,000 dalton subunit of eIF-3. Other initiation factors, eIF-4B and eIF-4A, and elongation factors, EF-1 and EF-2, are not phosphorylated by the kinase. Quantitative analysis indicates that the kinase catalyzes the incorporation of about 0.5 to 0.6 mole of phosphate per mole of the 42,000 dalton subunit of eIF-2 and about 6 moles of phosphate per mole of the 107,000 dalton subunit of eIF-3. Three proteins (M_r = 38,000, 14,800, and 12,600) of the 60S ribosomal subunit are phosphorylated by the kinase, but none of the 40S ribosomal proteins are substrates of the kinase. No effects of phosphorylation on the activities of eIF-2, eIF-3, or 60S ribosomal subunits could be demonstrated in vitro.