Interaction between non-formylated initiator Met-tRNA(fMet) and the ribosomal A-site from Escherichia coli

We report studies in vitro of the interaction between non-formylated initiator Met-tRNA(fMet) and 70S ribosomes. The binding of Met-tRNA(fMet) to ribosomes carrying fMet-tRNA(fMet) in the P-site is strongly stimulated by elongation factor EF-Tu:GTP in the presence of (AUG)3. The enzymatically bound Met-tRNA(fMet) does not react with puromycin. The bound Met-tRNA(fMet) can accept formylmethionine from P-site-bound fMet-tRNA(fMet). These results demonstrate a functionally active binding at the ribosomal A-site. Partial ribonuclease digestion (footprinting) was used to study the sites in Met-tRNA(fMet) which are involved in the interaction with the ribosomal A-site. The results show that a large part of the tRNA molecule is protected by the ribosome against ribonuclease digestion. In addition to the protection found in the amino acid region and the anticodon arm, protection is seen in the D-loop and in the extra arm. No region within the bound tRNA is found to be more accessible for RNases than in the free Met-tRNA(fMet). The reported enhancement of ribonuclease cuts in the D- and T-arms of A-site-bound Phe-tRNAPhe is thus not found in A-site bound Met-tRNA(fMet).     

Reactivity of the P-site-bound donor in ribosomal peptide-bond formation

The puromycin reaction, catalyzed by the ribosomal peptidyltransferase, has been carried out so as to make the definition of two distinct parameters of this reaction possible. These are (a) the final degree of the reaction which gives the proportion of peptidyl (P)-site binding of the donor and (b) the reactivity of the donor substrate expressed as an apparent rate constant (kobs). This kobs varies with the concentration of puromycin; the maximal value (k3) of the kobs, at saturating concentrations of puromycin, gives the reactivity of the donor independently of the concentrations of both the donor and puromycin. k3 is also a measure of the activity of peptidyltransferase expressed as its catalytic rate constant (kcat). If we assume that the puromycin-reactive donor is bound at the ribosomal P site, we observe the following, depending on the conditions of the experiment: the proportion of P-site binding of the donor substrates AcPhe-tRNA or fMet-tRNA can be the same and close to 100%, while there is a tenfold increase in the reactivity of the donor (k3 = 0.8 min-1 versus 8.3 min-1). On the other hand there are conditions, under which the proportion of P-site binding increases from 30% to 100% while k3 remains low and equal to 0.8 min-1. Using the puromycin reaction it was also found that an increase of Mg2+ from 10 mM to 20 mM reduces the reactivity of the donor and, hence, the activity of peptidyltransferase, provided that this change in Mg2+ occurs during the binding of the donor but not when it occurs during peptide bond formation per se. The fact that the donor substrate may exist in various states of reactivity in this cell-free system raises the possibility that the rate of peptide bond formation may not be uniform during protein synthesis.     

The A1 x U72 base pair conserved in eukaryotic initiator tRNAs is important specifically for binding to the eukaryotic translation initiation factor eIF2.

The formation of a specific ternary complex between eukaryotic initiation factor 2 (eIF2), the initiator methionyl-tRNA (Met-tRNA), and GTP is a critical step in translation initiation in the cytoplasmic protein-synthesizing system of eukaryotes. We show that the A1 x U72 base pair conserved at the end of the acceptor stem in eukaryotic and archaebacterial initiator methionine tRNAs plays an important role in this interaction. We changed the A1 x U72 base pair of the human initiator tRNA to G1 x C72 and expressed the wild-type and mutant tRNA genes in the yeast Saccharomyces cerevisiae by using constructs previously developed in our laboratory for expression of the human initiator tRNA gene in yeasts. We show that both the wild-type and mutant human initiator tRNAs are aminoacylated well in vivo. We have isolated the wild-type and mutant human initiator tRNAs in substantially pure form, free of the yeast initiator tRNA, and have analyzed their properties in vitro. The G1 x C72 mutation affects specifically the binding affinity of eIF2 for the initiator tRNA. It has no effect on the subsequent formation of 40S or 80S ribosome initiator Met-tRNA-AUG initiation complexes in vitro or on the puromycin reactivity of the Met-tRNA in the 80S initiation complex.     

The role of mammalian initiation factor eIF-4D and its hypusine modification in translation

Initiation factor eIF-4D functions late in the initiation pathway, apparently during formation of the first peptide bond. The factor is post-translationally modified at a specific lysine residue by reaction with spermidine and subsequent hydroxylation to form hypusine. A precursor form lacking hypusine is inactive in the assay for methionyl-puromycin synthesis, but activity is restored following in vitro modification to deoxyhypusine, thereby suggesting that the modification is essential for function. Since formylated methionyl-tRNA is less dependent on eIF-4D in the puromycin assay, we postulate that eIF-4D and its hypusine modification may stabilize charged Met-tRNA binding to the peptidyl transferase center of the 60S ribosomal subunit. Analysis of eIF-4D genes in yeast indicate that eIF-4D and its hypusine modification are essential for cell growth.     

Structural integrity of {alpha}-helix H12 in translation initiation factor eIF5B is critical for 80S complex stability

Translation initiation factor eIF5B promotes GTP-dependent ribosomal subunit joining in the final step of the translation initiation pathway. The protein resembles a chalice with the α-helix H12 forming the stem connecting the GTP-binding domain cup to the domain IV base. Helix H12 has been proposed to function as a rigid lever arm governing domain IV movements in response to nucleotide binding and as a molecular ruler fixing the distance between domain IV and the G domain of the factor. To investigate its function, helix H12 was lengthened or shortened by one or two turns. In addition, six consecutive residues in the helix were substituted by Gly to alter the helical rigidity. Whereas the mutations had minimal impacts on the factor's binding to the ribosome and its GTP binding and hydrolysis activities, shortening the helix by six residues impaired the rate of subunit joining in vitro and both this mutation and the Gly substitution mutation lowered the yield of Met-tRNA(i)(Met) bound to 80S complexes formed in the presence of nonhydrolyzable GTP. Thus, these two mutations, which impair yeast cell growth and enhance ribosome leaky scanning in vivo, impair the rate of formation and stability of the 80S product of subunit joining. These data support the notion that helix H12 functions as a ruler connecting the GTPase center of the ribosome to the P site where Met-tRNA(i)(Met) is bound and that helix H12 rigidity is required to stabilize Met-tRNA(i)(Met) binding.     

Mammalian translation initiation factor eIF1 functions with eIF1A and eIF3 in the formation of a stable 40 S preinitiation complex

We have examined the role of the mammalian initiation factor eIF1 in the formation of the 40 S preinitiation complex using in vitro binding of initiator Met-tRNA (as Met-tRNA(i).eIF2.GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA. We observed that, although both eIF1A and eIF3 are essential to generate a stable 40 S preinitiation complex, quantitative binding of the ternary complex to 40 S subunits also required eIF1. The 40 S preinitiation complex contained, in addition to eIF3, both eIF1 and eIF1A in a 1:1 stoichiometry with respect to the bound Met-tRNA(i). These three initiation factors also bind to free 40 S subunits, and the resulting complex can act as an acceptor of the ternary complex to form the 40 S preinitiation complex (40 S.eIF3.eIF1.eIF1A.Met-tRNA(i).eIF2.GTP). The stable association of eIF1 with 40 S subunits required the presence of eIF3. In contrast, the binding of eIF1A to free 40 S ribosomes as well as to the 40 S preinitiation complex was stabilized by the presence of both eIF1 and eIF3. These studies suggest that it is possible for eIF1 and eIF1A to bind the 40 S preinitiation complex prior to mRNA binding.     

Engaging the ribosome: universal IFs of translation

Eukaryotic initiation factor 1A (eIF1A) and the GTPase IF2/eIF5B are the only universally conserved translation initiation factors. Recent structural, biochemical and genetic data indicate that these two factors form an evolutionarily conserved structural and functional unit in translation initiation. Based on insights gathered from studies of the translation elongation factor GTPases, we propose that these factors occupy the aminoacyl-tRNA site (A site) on the ribosome, and promote initiator tRNA binding and ribosomal subunit joining. These processes yield a translationally competent ribosome with Met-tRNA in the ribosomal peptidyl-tRNA site (P site), base-paired to the AUG start codon of a mRNA.     

Inhibitors of protein synthesis V. Irreversible interaction of antibiotics with an initiation complex.

The initiation complex (t-complex) formed in a cell-free system (E. coli) from Ac-Phe-tRNA, poly(U) and washed ribosomes in the presence of initiation factors (ribosomal wash) and GTP, contains the Ac-Phe-tRNA bound quantitatively in a puromycin-reactive state. The t-complex is irreversibly inactivated by spiramycin with respect to its reactivity toward puromycin. The inactivated t-complex retains all of the Ac-Phe-tRNA bound, but it does not react with puromycin (2 x10-minus-3M) within 32 min at 25 degrees. In the case of another inhibitor protein synthesis, sparsomycin, the permanently "modified" t-complex not only retains all the bound Ac-Phe-tRNA but it can still react with puromycin. In the continuous presence of sparsomycin (1 x 10-minus-7M) the bound Ac-Phe-tRNA reacts quantitatively at a rate which is one-tenth the rate at which the t-complex reacts with puromycin, at low (6.25 x 10-minus-5M) or high (2 x 10-minus-3M) concentrations. These results are not in agreement with current views according to which aparsomycin binds to the ribosome reversibly at a single site with a KI in the range of 10-minus6-10-minus-7 M and according to which this stie is at the A'-site (puromycin site) of peptidyl transferase.     

Regulation of GTP hydrolysis prior to ribosomal AUG selection during eukaryotic translation initiation

Genetic studies in yeast have shown that the translation initiation factor eIF5 plays an important role in the selection of the AUG start codon. In order to ensure translation fidelity, the hydrolysis of GTP bound to the 40S preinitiation complex (40S.Met-tRNA(i).eIF2.GTP), promoted by eIF5, must occur only when the complex has selected the AUG start codon. However, the mechanism that prevents the eIF5-promoted GTP hydrolysis, prior to AUG selection by the ribosomal machinery, is not known. In this work, we show that the presence of initiation factors eIF1, eIF1A and eIF3 in the 40S preinitiation complex (40S.eIF1.eIF1A.eIF3.Met-tRNA(i).eIF2.GTP) and the subsequent binding of the preinitiation complex to eIF4F bound at the 5'-cap structure of mRNA are necessary for preventing eIF5-promoted hydrolysis of GTP in the 40S preinitiation complex. This block in GTP hydrolysis is released upon AUG selection by the 40S preinitiation complex. These results, taken together, demonstrate the biochemical requirements for regulation of GTP hydrolysis and its coupling to the AUG selection process during translation initiation.     

Eukaryotic initiation complex formation. Evidence for two distinct pathways.

Two distinct pathways have been elucidated which lead to the formation of an AUG-dependent initiation complex. One pathway involves the use of initiation factor M1 (IF-M1) to promote AUG-dependent binding of the initiator tRNA to the 40 S subunit, followed by joining of the 60 S subunit in the presence of IF-M2A, IF-M2B, and GTP. The second pathway involves the IF-MP-directed binding of initiator tRNA to the 40 S subunit via a ternary complex of IF-MP-GTP-Met-tRNAf. This reaction does not require AUG codon. However, subsequent formation of an 80 S initiation complex (as determined by methionyl-puromycin synthesis) required AUG as well as IF-M2A, IF-M2B, and GTP. Since both pathways require the same complementary initiation factors (at the same level), it would appear that the only difference is the manner in which the initiator tRNA is bound to the 40 S subunit, either by IF-M1 or IF-MP. Examination of the requirements for endogenous mRNA-directed methionyl-puromycin synthesis indicates a greater difference between IF-MP and IF-M1 in that only IF-MP was capable of forming an 80 S initiation complex which was sensitive to puromycin.     

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.     

Mutually exclusive binding of messenger RNA and initiator methionyl transfer RNA to eukaryotic initiation factor 2

Eukaryotic initiation factor 2 (eIF-2), purified to at least 98% homogeneity as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and containing no detectable amounts of eukaryotic initiation factor 4B (eIF-4B), is active both in the binding of Met-tRNA and in the binding of globin mRNA. The mRNA-binding activity is completely sensitive to competitive inhibition by Met-tRNA, provided GTP is present, but not by uncharged tRNA. By contrast, binding of mRNA to partially purified eIF-4B is not inhibited by Met-tRNA_f. These results establish that the only mRNA-binding component in the eIF-2 preparation is eIF-2 itself, and show that a given molecule of eIF-2 can either bind to a molecule of mRNA, or form a ternary complex with Met-tRNA_f and GTP, but cannot do both at once.     

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.     

Non-enzymic binding of formylmethionyl-transfer RNAf to Artemia salina ribosomes

40 S ribosomal subunits of Artemia salina embryos can bind formylmethionyl-transfer RNA_f non-enzymically, i.e., in the absence of initiation factors. This, like the enzymic reaction, is largely AUG-dependent. Much more fMet-tRNA_f is bound by 80 S ribosomes but, in this case, a large fraction (about two-thirds) of the binding is AUG-independent. Whereas the AUG-dependent binding is very sensitive to edeine, a potent initiation inhibitor, the AUG-independent binding is resistant to this antibiotic. Virtually all of the bound fMet-tRNA_f is in all cases capable of reacting with puromycin to form fMet-puromycin; hence the bound aminoaoyl-tRNA is in the peptidyl (donor) site of the 80 S ribosome. Non-acylated tRNAs also bind to this site with high affinity in a codon-independent reaction and block the 80 S binding of fMet-tRNA_f. The properties of the peptidyl site are consistent with a non-decoding site which harbors the initiator aminoacyl-tRNA, when the 80 S initiation complex is formed, and to which every molecule of tRNA remains temporarily attached following peptide bond synthesis.     

Analysis of the puromycin reaction. The ribosomal exclusion principle for AcPhe-tRNA binding re-examined

The standard technique for determination of the ribosomal site location of bound tRNA, viz. the puromycin reaction, has been analyzed with regard to its applicability under tRNA saturation conditions. The criteria derived have been used to re-examine the exclusion principle for peptidyl-tRNA binding, which states that only one peptidyl-tRNA (AcPhe-tRNA) can be bound per ribosome although in principle two sites (A and P site) are available. The following results were obtained. The puromycin reaction is only appropriate for a site determination if the reaction conditions prevent one ribosome from performing more than one puromycin reaction. With an excess of AcPhe-tRNA over ribosomes, and in the absence of EF-G, this criterion is fulfilled at 0 degree C, where the P-site-bound material reacts with puromycin (quantitative reaction after 50 h), while the A-site-bound material does not. In contrast, at 37 degrees C the extent of the puromycin reaction can exceed the binding values by 2-4-fold ('repetitive reaction'). In the presence of EF-G a repetitive puromycin reaction is seen even at 0 degree C, i.e. EF-G can already promote a translocation reaction at 0 degree C. However, the extent of translocation becomes negligibly low for short incubation times (up to 60 min) at 0 degree C, if only catalytic amounts of EF-G are used. Using the criteria outlined above, the validity of the exclusion principle for Escherichia coli ribosomes was confirmed pursuing two different experimental strategies. Ribosomes were saturated with AcPhe-tRNA at one molecule per 70S ribosome, and a quantitative puromycin reaction demonstrated the exclusive P-site location of the AcPhe-tRNA. The same result was also found in the presence of viomycin, which blocks the translocation reaction. These findings also indicate that here nearly 100% of the ribosomes participate in AcPhe-tRNA binding to the P site. Precharging the P sites of 70S ribosomes with one Ac[14C]Phe-tRNA molecule per ribosome prevented additional Ac[3H]Phe-tRNA binding. In contrast, 70S particles carrying one molecule of [14C]tRNAPhe per ribosome were able to bind up to a further 0.64 molecule Ac[3H]Phe-tRNA per ribosome.     

Localization of eukaryotic initiation factor 2 in neuron primary cultures and established cell lines

Eukaryotic initiation factor 2 (eIF-2) is a heterotrimeric protein with subunits α, β and γ that forms a ternary complex with Met-tRNA and GTP. It promotes the binding of Met-tRNA to ribosomes and controls translational rates via phosphorylation/dephosphorylation mechanisms. By means of immunofluorescence and post-embedding immunocytochemistry of intact cells and quantitative immunoblotting of cell extracts, the cellular distribution of the initiation factor has been examined in primary neuronal cultures as well as in two established cell lines: PC12 phaeochromocytoma cells and rat pituitary GH4C1 cells. Our results indicated that the initiation factor is located not only in the cytoplasm but also in the nuclei of the cultured neurons and cell lines. In the cytoplasm, immunocytochemical studies reveal that the factor is present mainly in those areas that are rich in ribosomes. In the nucleus, the immunolabelling of eukaryotic initiation factor 2 verified the presence of gold particles in both nucleolar and extranucleolar areas. The specific distribution of this factor on both sides of the nuclear envelope suggests that it might have some nuclear-related function(s) besides its already known role in the control of translation     

The crystal structure of the carboxy-terminal domain of human translation initiation factor eIF5

The carboxy-terminal domain (CTD) of eukaryotic initiation factor 5 (eIF5) plays a central role in the formation of the multifactor complex (MFC), an important intermediate for the 43 S pre-initiation complex assembly. The IF5-CTD interacts directly with the translation initiation factors eIF1, eIF2-beta, and eIF3c, thus forming together with eIF2 bound Met-tRNA(i)(Met) the MFC. In this work we present the high resolution crystal structure of eIF5-CTD. This domain of the protein is exclusively composed out of alpha-helices and is homologous to the carboxy-terminal domain of eIF2B-epsilon (eIF2Bepsilon-CTD). The most striking difference in the two structures is an additional carboxy-terminal helix in eIF5. The binding sites of eIF2-beta, eIF3 and eIF1 were mapped onto the structure. eIF2-beta and eIF3 bind to non-overlapping patches of negative and positive electrostatic potential, respectively.     

Localization of eukaryotic initiation factor 2 in neuron primary cultures and established cell lines

Eukaryotic initiation factor 2 (eIF-2) is a heterotrimeric protein with subunits α, β and γ that forms a ternary complex with Met-tRNA and GTP. It promotes the binding of Met-tRNA to ribosomes and controls translational rates via phosphorylation/dephosphorylation mechanisms. By means of immunofluorescence and post-embedding immunocytochemistry of intact cells and quantitative immunoblotting of cell extracts, the cellular distribution of the initiation factor has been examined in primary neuronal cultures as well as in two established cell lines: PC12 phaeochromocytoma cells and rat pituitary GH4C1 cells. Our results indicated that the initiation factor is located not only in the cytoplasm but also in the nuclei of the cultured neurons and cell lines. In the cytoplasm, immunocytochemical studies reveal that the factor is present mainly in those areas that are rich in ribosomes. In the nucleus, the immunolabelling of eukaryotic initiation factor 2 verified the presence of gold particles in both nucleolar and extranucleolar areas. The specific distribution of this factor on both sides of the nuclear envelope suggests that it might have some nuclear-related function(s) besides its already known role in the control of translation     

Purification and characterization of yeast mitochondrial initiation factor 2

Yeast mitochondrial initiation factor 2 (ymIF2) is encoded by the nuclear IFM1 gene. A His-tagged version of ymIF2, lacking its predicted mitochondrial presequence, was expressed in Escherichia coli and purified. Purified ymIF2 bound both E. coli fMet-tRNA(f)(Met) and Met-tRNA(f)(Met), but binding of formylated initiator tRNA was about four times higher than that of the unformylated species under the same conditions. In addition, the isolated ymIF2 was compared to E. coli IF2 in four other assays commonly used to characterize this initiation factor. Formylated and nonformylated Met-tRNA(f)(Met) were bound to E. coli 30S ribosomal subunits in the presence of ymIF2, GTP, and a short synthetic mRNA. The GTPase activity of ymIF2 was found to be dependent on the presence of E. coli ribosomes. The ymIF2 protected fMet-tRNA(f)(Met) to about the same extent as E. coli IF2 against nonenzymatic deaminoacylation. In contrast to E. coli IF2, the complex formed between ymIF2 and fMet-tRNA(f)(Met) was not stable enough to be analyzed in a gel shift assay. In similarity to other IF2 species isolated from bacteria or bovine mitochondria, the N-terminal domain could be eliminated without loss of initiator tRNA binding activity.