Eukaryotic Protein Synthesis Initiation Research Articles

Homeostasis in mRNA initiation: wheat germ poly(A)-binding protein lowers the activation energy barrier to initiation complex formation.

Previous kinetic binding studies of wheat germ protein synthesis eukaryotic initiation factor iso4F (eIFiso4F) and its subunit, eIF4E, with m(7)GTP and mRNA analogues indicated that binding occurred by a two-step process with the first step being too fast to measure by stopped-flow techniques (). Further equilibrium studies showed that poly(A)-binding protein (PABP) enhanced the cap binding of eIFiso4F about 40-fold. The kinetic effects of PABP on cap binding and the temperature dependence of this reaction were measured and compared. Fluorescence stopped-flow studies of the PABP.eIFiso4F protein complex with cap show a concentration-independent conformational change. PABP did not significantly increase the rate of the conformational change, and because the initial second-order binding is essentially diffusion-controlled, the enhancement of cap affinity must reside in the dissociation rate. The dissociation rate was more than 5-fold slower in the presence of PABP. The temperature dependence of the cap binding reaction was markedly reduced in the presence of PABP. The reduced energy barrier for formation of a cap.eIFiso4F complex suggests a more stable platform for further initiation complex formation and a possible means of adapting to varying temperature conditions.

Disruption of the Interaction of Mammalian Protein Synthesis Eukaryotic Initiation Factor 4B with the Poly(A)-binding Protein by Caspase- and Viral Protease-mediated Cleavages

Eukaryotic initiation factor (eIF) 4B interacts with several components of the initiation pathway and is targeted for cleavage during apoptosis. In a cell-free system, cleavage of eIF4B by caspase-3 coincides with a general inhibition of protein synthetic activity. Affinity chromatography demonstrates that mammalian eIF4B interacts with the poly(A)-binding protein and that a region consisting of the N-terminal 80 amino acids of eIF4B is both necessary and sufficient for such binding. This interaction is lost when eIF4B is cleaved by caspase-3, which removes the N-terminal 45 amino acids. Similarly, the association of eIF4B with the poly(A)-binding protein in vivo is reduced when cells are induced to undergo apoptosis. Cleavage of the poly(A)-binding protein itself, using human rhinovirus 3C protease, also eliminates the interaction with eIF4B. Thus, disruption of the association between mammalian eIF4B and the poly(A)-binding protein can occur during both apoptosis and picornaviral infection and is likely to contribute to the inhibition of translation observed under these conditions.

The kinase DYRK phosphorylates protein-synthesis initiation factor eIF2Bepsilon at Ser539 and the microtubule-associated protein tau at Thr212: potential role for DYRK as a glycogen synthase kinase 3-priming kinase.

The substrate specificity of glycogen synthase kinase 3 (GSK3) is unusual in that efficient phosphorylation only occurs if another phosphoserine or phosphothreonine residue is already present four residues C-terminal to the site of GSK3 phosphorylation. One such substrate is the epsilon-subunit of rat eukaryotic protein-synthesis initiation factor 2B (eIF2Bepsilon), which is inhibited by the GSK3-catalysed phosphorylation of Ser(535). There is evidence that GSK3 is only able to phosphorylate eIF2Bepsilon at Ser(535) if Ser(539) is already phosphorylated by another protein kinase. However, no protein kinases capable of phosphorylating Ser(539) have so far been identified. Here we show that Ser(539) of eIF2Bepsilon, which is followed by proline, is phosphorylated specifically by two isoforms of dual-specificity tyrosine phosphorylated and regulated kinase (DYRK2 and DYRK1A), but only weakly or not at all by other 'proline-directed' protein kinases tested. We also establish that phosphorylation of Ser(539) permits GSK3 to phosphorylate Ser(535) in vitro and that eIF2Bepsilon is highly phosphorylated at Ser(539) in vivo. The DYRK isoforms also phosphorylate human microtubule-associated protein tau at Thr(212) in vitro, a residue that is phosphorylated in foetal tau and hyperphosphorylated in filamentous tau from Alzheimer's-disease brain. Phosphorylation of Thr(212) primes tau for phosphorylation by GSK3 at Ser(208) in vitro, suggesting a more general role for DYRK isoforms in priming phosphorylation of GSK3 substrates.

Cell cycle arrest in archaea by the hypusination inhibitor N(1)-guanyl-1,7-diaminoheptane.

Hypusination is an essential posttranslational modification unique to archaeal and eukaryotic protein synthesis initiation factor 5A (aIF5A and eIF5A, respectively). We have investigated the effect of the efficient hypusination inhibitor N(1)-guanyl-1,7-diaminoheptane (GC(7)) on four archaeal and one bacterial species. We found that (i) archaea are sensitive to GC(7), whereas the bacterium Escherichia coli is not, (ii) GC(7) causes rapid and reversible arrest of growth of the archaeon Sulfolobus acidocaldarius, and (iii) the growth arrest is accompanied by a specific reversible arrest of the cell cycle prior to cell division. Our findings establish a link between hypusination and sustained growth of archaea and thereby provide the framework to study molecular details of archaeal cell cycle in connection with in vivo functions of hypusine and of aIF5A and eIF5A.

Changes in integrity and association of eukaryotic protein synthesis initiation factors during apoptosis.

Induction of apoptosis results in inhibition of the rate of overall protein synthesis in a variety of cell types. We have shown previously that polypeptide chain initiation factor eIF4GI is rapidly cleaved by caspase-3, whereas other components of the eIF4F complex are much more stable during apoptosis in BJAB and Jurkat cells. We have now extended our analysis to other factors involved in the initiation of protein synthesis and we report here that eIF4B, the p35 subunit of eIF3, and minor proportions of the alpha subunit of eIF2 and the eIF4E-binding protein 4E-BP1 are also cleaved to give rise to discrete fragments. These cleavages occur with delayed kinetics relative to that seen for eIF4GI, and eIF2beta and eIF2gamma levels also decrease at a relatively late stage of apoptosis. In contrast, the second form of eIF4G described recently, eIF4GII, is cleaved as rapidly as eIF4GI under the same conditions. Purified recombinant caspase-3 is able to degrade eIF4B and eIF3(p35) in vitro, producing fragments of the same sizes as those seen in intact cells. Induction of apoptosis also results in a biphasic change in the association of 4E-BP1 with eIF4E. Thus the progress of apoptosis is characterized by a complex programme of changes in several initiation factors, including the specific fragmentation or complete degradation of some and alterations in the association status of others. These events are likely to contribute to the inhibition of protein synthesis seen under these conditions.

The joining of ribosomal subunits in eukaryotes requires eIF5B.

Initiation of eukaryotic protein synthesis begins with the ribosome separated into its 40S and 60S subunits. The 40S subunit first binds eukaryotic initiation factor (eIF) 3 and an eIF2-GTP-initiator transfer RNA ternary complex. The resulting complex requires eIF1, eIF1A, eIF4A, eIF4B and eIF4F to bind to a messenger RNA and to scan to the initiation codon. eIF5 stimulates hydrolysis of eIF2-bound GTP and eIF2 is released from the 48S complex formed at the initiation codon before it is joined by a 60S subunit to form an active 80S ribosome. Here we show that hydrolysis of eIF2-bound GTP induced by eIF5 in 48S complexes is necessary but not sufficient for the subunits to join. A second factor termed eIF5B (relative molecular mass 175,000) is essential for this process. It is a homologue of the prokaryotic initiation factor IF2 (re and, like it, mediates joining of subunits and has a ribosome-dependent GTPase activity that is essential for its function.

Analysis of surgical margins with the molecular marker eIF4E: a prognostic factor in patients with head and neck cancer.

Complete excision of cancer is guided by histologic assessment of surgical margins. Molecular markers may be more sensitive in identifying malignant cells. eIF4E, a eukaryotic protein synthesis initiation factor, is found elevated in all head and neck squamous cell cancers (HNSCC). In a preliminary study using Western blots and a retrospective study using immunohistochemistry, eIF4E elevation in histologically tumor-free surgical margins correlated with a higher local-regional recurrence. We wanted to confirm this hypothesis in a prospective study. Immunohistochemical analysis of surgical margins and tumors with an antibody to eIF4E was performed on all newly diagnosed HNSCC patients who underwent surgical resection for their disease between January 1996 and December 1997. All 65 patients had elevated levels of eIF4E in the tumors. Thirty-six patients (55%) had elevated eIF4E in histologically tumor-free margins, and 20 of these patients (56%) have had local-regional recu rrences. Twenty-nine patients (45%) had no elevation of eIF4E in the margins, and only two of these patients (6.9%) have had recurrences. Cox regression analysis showed that elevated eIF4E in the margins was an independent prognostic factor (P =.009) for recurrence. The Kaplan-Meier curves for the probability of nonrecurrence were significantly different for positive and negative eIF4E margins (P =. 0001, log-rank test). In histologically tumor-free surgical margins, elevated levels of eIF4E predict a significantly increased risk of recurrence. Elevated levels of eIF4E in tumor margins may identify patients who could benefit from additional therapy.

Five different genes, Eif4a1, Cd68, Supl15h, Sox15 and Fxr2h, are clustered in a 40 kb region of mouse chromosome 11.

We characterized a region of the mouse genome disrupted by integration of a gene trap (GT) vector in ES cells. On 5' rapid amplification of cDNA ends analysis of the fusion transcripts containing the GT vector, we identified the eukaryotic protein synthesis initiation factor 4A1 gene (Eif4a1) as a promoter-trapped gene. Plasmid rescue was used to show that the other end of the integrated vector disrupted the murine homolog of the human fragile X mental retardation syndrome-related protein 2 gene (Fxr2h). Structural analysis of P1 clones, isolated from the wild-type mouse genome by PCR with Eif4a1-specific primers, indicated that the integration of the GT vector was accompanied by the deletion of about 35 kb of genomic DNA and that the disrupted region also included three genes, Cd68, Supl15h and Sox15, the latter two of which are transcribed in opposite directions with overlapping 3' ends. These five different genes at least, Eif4a1, Cd68, Supl15h, Sox15 and Fxr2h, are clustered in a 40 kb region. The chromosomal location of this region was mapped by means of interspecific backcross panel DNAs to the central part of mouse chromosome 11, exhibiting a known region of synteny with human chromosome 17.

Caspase-3 is necessary and sufficient for cleavage of protein synthesis eukaryotic initiation factor 4G during apoptosis

Induction of apoptosis BJAB cells is accompanied by the rapid cleavage of protein synthesis eukaryotic initiation factor 4G and the appearance of a fragment of approximately 76 kDa. Inhibition of apoptotic proteases (caspases) has previously been shown to prevent the cleavage of eukaryotic initiation factor 4G. In MCF-7 breast carcinoma cells, which are deficient in caspase-3, eukaryotic initiation factor 4G is not cleaved but in vivo expression of caspase-3 restores eukaryotic initiation factor 4G cleavage following induction of apoptosis. Recombinant caspase-3 can also cleave eukaryotic initiation factor 4G to yield the 76 kDa fragment both in cell extracts and when the eukaryotic initiation factor 4G is presented in a purified eukaryotic initiation factor 4F complex. These results indicate that caspase-3 activity is necessary and sufficient for eukaryotic initiation factor 4G degradation.

Multiple Isoforms of Eukaryotic Protein Synthesis Initiation Factor 4E in Caenorhabditis elegans Can Distinguish between Mono- and Trimethylated mRNA Cap Structures

The rate-limiting step for cap-dependent translation initiation in eukaryotes is recruitment of mRNA to the ribosome. An early event in this process is recognition of the m7GTP-containing cap structure at the 5'-end of the mRNA by initiation factor eIF4E. In the nematode Caenorhabditis elegans, mRNAs from 70% of the genes contain a different cap structure, m32,2,7GTP. This cap structure is poorly recognized by mammalian elF4E, suggesting that C. elegans may possess a specialized form of elF4E that can recognize m32,2,7GTP. Analysis of the C. elegans genomic sequence data base revealed the presence of three elF4E-like genes, here named ife-1, ife-2, and ife-3. cDNAs for these three eIF4E isoforms were cloned and sequenced. Isoform-specific antibodies were prepared from synthetic peptides based on nonhomologous regions of the three proteins. All three eIF4E isoforms were detected in extracts of C. elegans and were retained on m7GTP-Sepharose. One eIF4E isoform, IFE-1, was also retained on m32,2,7GTP-Sepharose. Furthermore, binding of IFE-1 and IFE-2 to m7GTP-Sepharose was inhibited by m32,2,7GTP. These results suggest that IFE-1 and IFE-2 bind both m7GTP- and m32,2, 7GTP-containing mRNA cap structures, although with different affinities. In conjunction with IFE-3, these eIF4E isoforms would permit cap-dependent recruitment of all C. elegans mRNAs to the ribosome.

EIF2 independently binds two distinct eIF2B subcomplexes that catalyze and regulate guanine-nucleotide exchange.

eIF2B is a heteropentameric guanine-nucleotide exchange factor essential for protein synthesis initiation in eukaryotes. Its activity is inhibited in response to starvation or stress by phosphorylation of the alpha subunit of its substrate, translation initiation factor eIF2, resulting in reduced rates of translation and cell growth. We have used an in vitro nucleotide-exchange assay to show that wild-type yeast eIF2B is inhibited by phosphorylated eIF2 [eIF2(alphaP)] and to characterize eIF2B regulatory mutations that render translation initiation insensitive to eIF2 phosphorylation in vivo. Unlike wild-type eIF2B, eIF2B complexes with mutated GCN3 or GCD7 subunits efficiently catalyzed GDP exchange using eIF2(alphaP) as a substrate. Using an affinity-binding assay, we show that an eIF2B subcomplex of the GCN3, GCD7, and GCD2 subunits binds to eIF2 and has a higher affinity for eIF2(alphaP), but it lacks nucleotide-exchange activity. In contrast, the GCD1 and GCD6 subunits form an eIF2B subcomplex that binds equally to eIF2 and eIF2(alphaP). Remarkably, this second subcomplex has higher nucleotide-exchange activity than wild-type eIF2B that is not inhibited by eIF2(alphaP). The identification of regulatory and catalytic eIF2B subcomplexes leads us to propose that binding of eIF2(alphaP) to the regulatory subcomplex prevents a productive interaction with the catalytic subcomplex, thereby inhibiting nucleotide exchange.

An alpha subunit-deficient form of eukaryotic protein synthesis initiation factor eIF-2 from rabbit reticulocyte lysate and its activity in ternary complex formation.

Eukaryotic protein synthesis initiation factor eIF-2 is usually isolated as a heterotrimer (alphabeta gamma). By use of Sephacryl S-300 fractionation an alpha subunit-deficient form of eIF-2 was identified in impure preparations from rabbit reticulocyte lysate and it appeared in these preparations to be still active in formation of the ternary complex (eIF-2.GTP.Met-tRNAi). Subsequently alpha subunit-deficient eIF-2 was further purified and this appeared to have retained ternary complex forming activity. Together with a suggested lack of involvement of the beta subunit this implies that the alpha subunit was not required for activity and the gamma subunit bound both GTP and Met-tRNAi in formation of the ternary complex. The identification and study of alpha subunit-deficient eIF-2 thus elucidated the involvement of the subunits in binding of GTP and Met-tRNAi to produce the ternary complex in polypeptide chain initiation.

Identification of the cap binding domain of human recombinant eukaryotic protein synthesis initiation factor 4E using a photoaffinity analogue.

Binding of eIF-4E to the 5' m7G cap structure of eukaryotic mRNA signals the initiation of protein synthesis. In order to investigate the molecular basis for this recognition, photoaffinity labeling with [gamma-32P]8-N3GTP was used in binding site studies of human recombinant cap binding protein eIF-4E. Competitive inhibition of this cap analogue by m7GTP and capped mRNA indicated probe specificity for interaction at the protein binding site. Saturation of the binding site with [gamma-32P]8-N3GTP further demonstrated the selectivity of photoinsertion. Aluminum (III)-chelate chromatography and reverse-phase HPLC were used to isolate the binding site peptide resulting from digestion of photolabeled eIF-4E with modified trypsin. Amino acid sequencing identified the binding domain as the region containing the sequence Trp 113-Arg 122.Lys 119 was not identified in sequencing analysis nor was it cleaved by trypsin. These results indicate that Lys 119 is the residue directly modified by photoinsertion of [gamma-32P]8-N3GTP. A detailed understanding of eIF-4E.m7G mRNA cap interactions may lead the way to regulating this essential protein-RNA interaction for specific mRNA in vivo.

Non-Ideal Size Exclusion Chromatography of Eukaryotic Protein Synthesis Initiation Factor 2

Eukaryotic protein synthesis initiation factor 2 (eIF-2), a heterotrimer, was chromatographed on high performance (Superose 6) and standard (Ultrogel AcA 34 and Sephacryl S-300) size exclusion chromatography (SEC) media. The retention volume of eIF-2 on the Superose 6 column was higher than expected from its Mr and elution of a βγ eIF-2 dimer was seen. Conversely, on AcA 34 and Sephacryl S-300 most of the eIF-2 was eluted at its expected position, considering its ellipsoidal structure, as the heterotrimer. Elution of the βγ heterodimeric eIF-2 may therefore be promoted by Superose 6 high performance SEC which therefore provides a means for its rapid preparation for studies in protein synthesis initiation.

The 58-kDa Cellular Inhibitor of the Double Stranded RNA-dependent Protein Kinase Requires the Tetratricopeptide Repeat 6 and DnaJ Motifs to Stimulate Protein Synthesis in Vivo

Double stranded RNA-dependent protein kinase (PKR) is a double stranded RNA-activated, interferon-induced serine-threonine kinase that participates in both the antiviral and antiproliferative properties of interferon. We previously found that influenza virus inhibited PKR function by recruiting or activating a cellular inhibitor termed P58(IPK). The present study was undertaken to complement our earlier analyses, which demonstrated that P58(IPK) efficiently inhibited PKR autophosphorylation and activity in vitro. We now report that P58(IPK) down-regulates PKR and, in turn, stimulates protein synthetic rates inside the cell. Using transfection analysis, we show that P58(IPK) stimulates translation of secreted embryonic alkaline phosphatase reporter gene mRNA. Furthermore, we found that at least two regions of the P58(IPK) molecule were required for PKR inhibitory activity in COS-1 cells: (i) the DnaJ similarity region at the carboxyl terminus (amino acids 391-504); and (ii) the tetratricopeptide repeat 6 (TPR6) domain (amino acids 222-255) located in the middle of the P58(IPK) protein and within the eukaryotic protein synthesis initiation factor 2alpha homology region. P58(IPK) variants lacking either one of these regions were unable to stimulate secreted embryonic alkaline phosphatase protein synthetic rates. Consistent with this data is the observation that the DeltaTPR6 mutant (the P58(IPK) variant lacking the TPR6 motif) failed to block PKR activity in vitro. Based on these data and our earlier in vitro functional and PKR-P58(IPK) binding analyses, a revised model of PKR regulation by P58(IPK) is presented.

The C-terminal domain of eukaryotic protein synthesis initiation factor (eIF) 4G is sufficient to support cap-independent translation in the absence of eIF4E.

The foot and mouth disease virus, a picornavirus, encodes two forms of a cysteine proteinase (leader or L protease) that bisects the EIF4G polypeptide of the initiation factor complex eIF4F into N-terminal (Nt) and C-terminal (Ct) domains. Previously we showed that, although in vitro cleavage of the translation initiation factor, eIF4G, with L protease decreases cap-dependent translation, the cleavage products themselves may directly promote cap-dependent protein synthesis. We now demonstrate that translation of uncapped mRNAs normally exhibits a strong requirement for eIF4F. However, this dependence is abolished when eIF4G is cleaved, with the Ct domain capable of supporting translation in the absence of the Nt domain. In contrast, the efficient translation of the second cistron of bicistronic mRNAs, directed by two distinct Internal Ribosome Entry Segments (IRES), exhibits no requirement for eIF4E but is dependent upon either intact eIF4G or the Ct domain. These results demonstrate that: (i) the apparent requirement for eIF4F for internal initiation on IRES-driven mRNAs can be fulfilled by the Ct proteolytic cleavage product; (ii) when eIF4G is cleaved, the Ct domain can also support cap-independent translation of cellular mRNAs not possessing an IRES element, in the absence of eIF4E; and (iii) when eIF4G is intact, translation of cellular mRNAs, whether capped or uncapped, is strictly dependent upon eIF4E. These data complement recent work in other laboratories defining the binding sites for other initiation factors on the eIF4G molecule.

Mapping of Functional Domains in Eukaryotic Protein Synthesis Initiation Factor 4G (eIF4G) with Picornaviral Proteases: IMPLICATIONS FOR CAP-DEPENDENT AND CAP-INDEPENDENT TRANSLATIONAL INITIATION

Cap-dependent binding of mRNA to the 40 S ribosomal subunit during translational initiation requires the association of eukaryotic initiation factor 4G (eIF4G; formerly eIF-4 gamma and p220) with other initiation factors, notably eIF4E, eIF4A, and eIF3. Infection of cells by picornaviruses results in proteolytic cleavage of eIF4G and generation of a cap-independent translational state. Rhinovirus 2A protease and foot-and-mouth-disease virus L protease were used to analyze the association of eIF4G with eIF4A, eIF4E, and eIF3. Both proteases bisect eIF4G into N- and C-terminal fragments termed cpN and cpC. cpN was shown to contain the eIF4E-binding site, as judged by retention on m7GTP-Sepharose, whereas cpC was bound to eIF3 and eIF4A, based on ultracentrifugal co-sedimentation. Further proteolysis of cpN by L protease produced an 18-kDa polypeptide termed cpN2 which retained eIF4E binding activity and corresponded to amino acid residues 319-479 of rabbit eIF4G. Further proteolysis of cpC yielded several smaller fragments. cpC2 (approximately 887-1402) contained the eIF4A binding site, whereas cpC3 (approximately 480-886) contained the eIF3 binding site. These results suggest that cleavage by picornaviral proteases at residues 479-486 separates eIF4G into two domains, one required for recruiting capped mRNAs and one for attaching mRNA to the ribosome and directing helicase activity. Only the latter would appear to be necessary for internal initiation of picornaviral RNAs.

Phosphorylation of Eukaryotic Protein Synthesis Initiation Factor 4E by Insulin-stimulated Protamine Kinase

Insulin-stimulated protamine kinase (cPK) and protein kinase C (PKC) phosphorylated eukaryotic protein synthesis initiation factor 4E (eIF-4E) on serine and threonine residues located on an identical tryptic fragment as judged by two-dimensional phosphopeptide mapping. With cPK and PKC, the apparent Km for eIF-4E was about 1.2 and 50 microM, respectively. Relative to recombinant human eIF-4E, cPK exhibited about 100% and < or = 5% activity with eIF-4ES209A and eIF-4ET210A, respectively, and eIF-4ES209A was phosphorylated exclusively on threonines. Bovine kidney eIF-4E enhanced up to 1.8-fold globin synthesis in m7GTP-Sepharose-treated reticulocyte lysates. In contrast, following incubation with cPK, these eIF-4E preparations stimulated globin synthesis up to 6-fold. Compared to the dephosphorylation of the cPK-modified serine on eIF-4E, reticulocyte lysates and highly purified protein phosphatase 2A exhibited marked preference for the cPK-modified threonine. The results indicate that cPK phosphorylates eIF-4E on Ser209 and Thr210, that the hydroxyl group or phosphorylation of Thr210 is necessary for cPK to act on Ser209, and that Ser209 phosphorylation activates reticulocyte globin synthesis. The results suggest that cPK could contribute to the insulin-stimulated phosphorylation of eIF-4E, but that protein phosphatase 2A may confer the site specificity of this response.

Phosphorylation of Eukaryotic Protein Synthesis Initiation Factor 4E at Ser-209

Initiation factor 4E (eIF-4E) binds to the m7GTP-containing cap of eukaryotic mRNA and facilitates the entry of mRNA into the initiation cycle of protein synthesis. eIF-4E is a phosphoprotein, and the phosphorylated form binds to mRNA caps 3-4-fold more tightly than the nonphosphorylated form. A previous study indicated that the major phosphorylation site was Ser-53 (Rychlik, W., Russ, M. A., and Rhoads, R. E. (1987) J. Biol. Chem. 262, 10434-10437). In the present study, we synthesized the phosphopeptide expected to result from tryptic digestion of eIF-4E, O-phosphoseryllysine. Surprisingly, the tryptic and synthetic phosphopeptides did not comigrate electrophoretically. Accordingly, we redetermined the phosphorylation site by isolating a chymotryptic phosphopeptide on reverse phase high performance liquid chromatography. The peptide was sequenced by Edman degradation and corresponded to 198QSHADTATKSGSTTKNRF215. The site of phosphorylation was determined to be Ser-209 by four methods: the increase in the ratio of dehydroalanine to serine derivatives during Edman degradation, the release of 32P, the further digestion of the chymotryptic phosphopeptide with trypsin, Glu-C, and Asp-N, and site-directed mutagenesis of eIF-4E cDNA. The S209A variant was not phosphorylated in a rabbit reticulocyte lysate system, whereas the wild-type, S53A, and S207A variants were. This site falls within the consensus sequence for phosphorylation by protein kinase C.

Thermodynamic characterization of the cooperativity of 40S complex formation during the initiation of eukaryotic protein synthesis.

The first step in mammalian protein synthesis is the formation of the 40S initiation complex, composed of the 40S ribosomal subunit (R), mRNA (M, here, a 10-mer oligoribonucleotide analogue containing the initiation codon), and the quaternary complex (Q, composed of eIF-2, GTP, Met-tRNA(fMet), and the ancillary protein factor Co-eIF-2C). The interdependence of the binding of R, M, and Q in forming the 40S complex is currently unclear. We have determined the thermodynamic parameters that characterize these interactions. The binary constants for R+M and Q+M were determined spectroscopically, measuring changes in the anisotropy of the fluorescence emission of 3'-fluorescein labeled M. The other binary constant, for Q+R, and the ternary constant were determined from Millipore filtration assays using radiolabeled Met-tRNA(fMet). The association constants for the binary reactions were as follows: Ka(Q,M) < or = 0.14 x 10(6) M-1, Ka(R,M) = 1.78 x 10(6) M-1, and Ka(Q,R) = 0.94 x 10(6) M-1. The binding of Q to R.M was markedly greater than that of Q to R [Ka(Q,R.M)/Ka(Q,R) > 62]. High cooperativity for this interaction occurs in either a single-site model or in lattice models for the binding of M to R. Data obtained using five other RNA 10-mers, each with the sequence altered at the AUG codon, suggest that this cooperativity is AUG dependent. The data are consistent with a scheme in which mRNA and Q bind independently to the 40S ribosome, but when the AUG codon is properly aligned with Q, a conformational change results in a 2.4 kcal/mol stabilization of the complex.