Translation initiation factor (eIF) 4B affects the rates of binding of the mRNA m7G cap analogue to wheat germ eIFiso4F and eIFiso4F.PABP

Previous kinetic binding studies of wheat germ protein synthesis eukaryotic translational initiation factor eIFiso4F and its subunit, eIFiso4E, with m(7)GTP and mRNA analogues indicated that binding occurred by a two-step process with the first step occurring at a rate close to the diffusion-controlled rate [Sha, M., Wang, Y., Xiang, T., van Heerden, A., Browning, K. S., and Goss, D. J. (1995) J. Biol. Chem. 270, 29904-29909]. The kinetic effects of eIF4B, PABP, and wheat germ eIFiso4F with two mRNA cap analogues and the temperature dependence of this reaction were measured and compared. The Arrhenius activation energies for binding of the two mRNA cap analogues, Ant-m(7)GTP and m(7)GpppG, were significantly different. Fluorescence stopped-flow studies of the eIFiso4F.eIF4B protein complex with two m(7)G cap analogues show a concentration-independent conformational change. The rate of this conformational change was approximately 2.4-fold faster for the eIFiso4F.eIF4B complex compared with our previous studies of eIFiso4F [Sha, M., Wang, Y., Xiang, T., van Heerden, A., Browning, K. S., and Goss, D. J. (1995) J. Biol. Chem. 270, 29904-29909]. The dissociation rates were 3.7- and 5.4-fold slower for eIFiso4F.Ant-m(7)GTP and eIFiso4F.m(7)GpppG, respectively, in the presence of eIF4B and PABP. These studies show that eIF4B and PABP enhance the interaction with the cap and probably are involved in protein-protein interactions as well. The temperature dependence of the cap binding reaction was markedly reduced in the presence of either eIF4B or PABP. However, when both eIF4B and PABP were present, not only was the energy barrier reduced but the binding rate was faster. Since cap binding is thought to be the rate-limiting step in protein synthesis, these two proteins may perform a critical function in regulation of the overall protein synthesis efficiency. This suggests that the presence of both proteins leads to a rapid, stable complex, which serves as a scaffold for further initiation complex formation.     

Formation of ribosome-RNA initiation complexes with alfalfa mosaic virus RNA 4 and RNA 3.

RNA 4 of alfalfa mosaic virus (AMV) is a monocistronic messenger for the coat protein. We have determined the sequence of the 40 +/- 2 nucleotides in RNA 4 that were protected in the initiation complex formed with wheat germ 80 S ribosomes from digestion by T1 or pancreatic ribonucleases. The AUG coat protein initiation codon was near the middle of this protected region. We have found two ribosome-binding sites in RNA 3. The principal one, near the 5' end, is the initiation site for the major translation product, a 35,000 dalton protein. The second site binds ribosomes only weakly, at the beginning of the "silent" coat protein cistron, and is similar but not identical to the initiation site protection site is discussed.     

The yeast nuclear cap binding complex can interact with translation factor eIF4G and mediate translation initiation

The mRNA cap structure is bound by either the nuclear (CBC) or the cytoplasmic (eIF4F) cap binding complex. Following mRNA export, CBC must be exchanged for eIF4F in the cytoplasm. It is not known how this exchange occurs or how this RNP remodeling event is integrated with mRNA function. Here we report genetic and biochemical evidence that the yeast translation initiation factor eIF4G associates with CBC, and that eIF4E, the eIF4F component that binds both the cap and eIF4G, antagonizes this interaction. Furthermore, we find that CBC can stimulate translation in extracts containing an eIF4G protein deficient for eIF4E binding. These data suggest that eIF4E binding to the eIF4G-CBC complex on newly exported mRNA displaces CBC, and that the first round of translation on mRNA may occur via a different mechanism than subsequent rounds.     

Thermodynamics of mRNA 5' cap binding by eukaryotic translation initiation factor eIF4E

Translation of mRNA in eukaryotes begins with specific recognition of the 5' cap structure by the highly conserved protein, eIF4E. The thermodynamics of eIF4E interaction with nine chemical cap analogues has been studied by means of emission spectroscopy. High-sensitivity measurements of intrinsic protein fluorescence quenching upon cap binding provided equilibrium association constants in the temperature range of 279 to 314 K. A van't Hoff analysis yielded the negative binding enthalpies for the entire cap analogue series, -16.6 to -81 kJ mol(-1), and the entropies covering the range of +40.3 to -136 J mol(-1) K(-1) at 293 K. The main enthalpic contributions come from interactions of the phosphate chains and positively charged amino acids and the cation-pi stacking of 7-methylguanine with tryptophans. A nontrivial, statistically important isothermal enthalpy-entropy compensation has been detected (T(c) = 399 +/- 24 K), which points to significant fluctuations of apo-eIF4E and indicates that the cap-binding microstate lies 9.66 +/- 1.7 kJ mol(-1) below the mean energy of all available conformational states. For five cap analogues, large and positive heat capacity changes have been found. The values of DeltaC(p) degrees correlate with the free energies of eIF4E binding due to stiffening of the protein upon interaction with cap analogues. At biological temperatures, binding of the natural caps has both favorable enthalpy and favorable entropy. Thermodynamic coupling of cap-eIF4E association to intramolecular self-stacking of dinucleotide cap analogues strongly influences the enthalpies and entropies of the binding, but has a negligible effect on the resultant DeltaG degrees and DeltaC(p) degrees values.     

The eukaryotic translation initiation factor 4E controls lettuce susceptibility to the Potyvirus Lettuce mosaic virus

The eIF4E and eIF(iso)4E cDNAs from several genotypes of lettuce (Lactuca sativa) that are susceptible, tolerant, or resistant to infection by Lettuce mosaic virus (LMV; genus Potyvirus) were cloned and sequenced. Although Ls-eIF(iso)4E was monomorphic in sequence, three types of Ls-eIF4E differed by point sequence variations, and a short in-frame deletion in one of them. The amino acid variations specific to Ls-eIF4E(1) and Ls-eIF4E(2) were predicted to be located near the cap recognition pocket in a homology-based tridimensional protein model. In 19 lettuce genotypes, including two near-isogenic pairs, there was a strict correlation between these three allelic types and the presence or absence of the recessive LMV resistance genes mo1(1) and mo1(2). Ls-eIF4E(1) and mo1(1) cosegregated in the progeny of two separate crosses between susceptible genotypes and an mo1(1) genotype. Finally, transient ectopic expression of Ls-eIF4E restored systemic accumulation of a green fluorescent protein-tagged LMV in LMV-resistant mo1(2) plants and a recombinant LMV expressing Ls-eIF4E degrees from its genome, but not Ls-eIF4E(1) or Ls-eIF(iso)4E, accumulated and produced symptoms in mo1(1) or mo1(2) genotypes. Therefore, sequence correlation, tight genetic linkage, and functional complementation strongly suggest that eIF4E plays a role in the LMV cycle in lettuce and that mo1(1) and mo1(2) are alleles coding for forms of eIF4E unable or less effective to fulfill this role. More generally, the isoforms of eIF4E appear to be host factors involved in the cycle of potyviruses in plants, probably through a general mechanism yet to be clarified.     

Structural requirements for the specific recognition of an m7G mRNA cap

7-Methylguanosine (m(7)G), also known as the mRNA "cap", is used as a molecular tag in eukaryotic cells to mark the 5' end of messenger RNAs. The mRNA cap is required for several key events in gene expression in which the m(7)G moiety is specifically recognized by cellular proteins. The configurations of the m(7)G-binding pockets of a cellular (eIF4E) and a viral (VP39) cap-binding protein have been determined by X-ray crystallography. The binding energy has been hypothesized to result from a pi-pi stacking interaction between aromatic residues sandwiching the m(7)G base in addition to hydrogen bonds between the base and acidic protein side chains. To further understand the structural requirements for the specific recognition of an m(7)G mRNA cap, we determined the effects of amino acid substitutions in eIF4E and VP39 cap-binding sites on their affinity for m(7)GDP. The requirements for residues suggested to pi-pi stack and hydrogen bond with the m(7)G base were examined in each protein by measuring their affinities for m(7)GDP by fluorimetry. The results suggest that both eIF4E and VP39 require a complicated pattern of both orientation and identity of the stacking aromatic residues to permit the selective binding of m(7)GDP.     

Influence of 5'-terminal cap structure on the initiation of translation of vaccinia virus mRNA

The ability of methylated vaccinia virus mRNA to bind to ribosomes derived from wheat germ of rabbit reticulocyte lysates has been studied after beta elimination, to remove the 5'-terminal m7G, and after "recapping" of beta-eliminated mRNA molecules using guanylyltransferase.guanine-7-methyltransferase complex from vaccinia virions. Removal of m7G from the mRNA results in significant loss of ability to bind to ribosomes and to simulate protein synthesis in vitro. Readdition of m7G, but not of unmethylated guanosine to the 5' end results in recovery of both of these functions. To evaluate the role of 2'-O-methylation of the penultimate ribonucleoside, mRNAs containing m7G-(5')pppA- and m7G(5')pppG- as well as m7G(5')pppAm- and m7G(5')pppGm- ends were synthesized in vitro at limiting S-adenosylmethionine concentrations by vaccinia virus cores. By comparing the cap sequences of ribosome-bound and unbound mRNAs, we concluded that 2'-O-methylation has at most a minor effect compared to that of m7G upon ribosome binding under in vitro conditions. Only at high input mRNA concentrations, at which competition might occur, was there some ribodomal enrichment of mRNAs containing a specific terminal structure, namely m7G(5')pppAm-.     

RNA aptamers to mammalian initiation factor 4G inhibit cap-dependent translation by blocking the formation of initiation factor complexes

Eukaryotic translation initiation factor 4G (eIF4G) plays a crucial multimodulatory role in mRNA translation and decay by interacting with other translation factors and mRNA-associated proteins. In this study, we isolated eight different RNA aptamers with high affinity to mammalian eIF4G by in vitro RNA selection amplification. Of these, three aptamers (apt3, apt4, and apt5) inhibited the cap-dependent translation of two independent mRNAs in a rabbit reticulocyte lysate system. The cap-independent translation directed by an HCV internal ribosome entry site was not affected. Addition of exogenous eIF4G reversed the aptamer-mediated inhibition of translation. Even though apt3 and apt4 were selected independently, they differ only by two nucleotides. The use of truncated eIF4G variants in binding experiments indicated that apt4 (and probably apt3) bind to both the middle and C-terminal domains of eIF4G, while apt5 binds only to the middle domain of eIF4G. Corresponding to the difference in the binding sites in eIF4G, apt4, but not apt5, hindered eIF4G from binding to eIF4A and eIF3, in a purified protein solution system as well as in a crude lysate system. Therefore, the inhibition of translation by apt4 (and apt3) is due to the inhibition of formation of initiation factor complexes involving eIF4A and eIF3. On the other hand, apt5 had a much weaker affinity to eIF4G than apt4, but inhibited translation much more efficiently by an unknown mechanism. The five additional aptamers have sequences and predicted secondary structures that are largely different from each other and from apt3 through apt5. Therefore, we speculate that these seven sets of aptamers may bind to different regions in eIF4G in different fashions.     

Kinetics of binding the mRNA cap analogues to the translation initiation factor eIF4E under second-order reaction conditions

The kinetics of binding of five analogues of the 5'-mRNA cap, differing in size and electric charge, to the eukaryotic initiation factor eIF4E, at 20 degrees C, pH 7.2, and ionic strength of 150 mM, were measured, after mixing solutions of comparable concentrations of the reagents, in a stopped-flow spectrofluorimeter. The registered stopped-flow signals were fitted using an efficient software package, called Dyna Fit, based on a numerical solution of the kinetic rate equations for assumed reaction mechanisms. One-, two-, and three-step binding models were considered. The quality of fits for these models were compared using two statistical criteria: Akaike's Information Criterion and Bayesian Information Criterion. Based on resulting probabilities of the models, it was concluded that for all investigated ligands a one-step binding model has essentially no support in the experimental observations. Our conclusions were also analysed from the perspective of kinetic transients obtained for cap-eIF4E systems under the so called pseudo-first order reaction condition, which result in the linear correlation of the observed association rate constant with ligand concentration. The existence of such a linear correlation is usually considered as proof of a one-step binding mechanism. The kinetic and optical parameters, derived from fitting a two-step cap-binding model with the DynaFit, were used to simulate kinetic transients under pseudo-first order reaction conditions. It appeared that the observed association rate constants derived from these simulated transients are also linearly correlated with the ligand concentration. This indicated that these linear dependencies are not sufficient to conclude a one-step binding.     

Characterization of pokeweed antiviral protein binding to mRNA cap analogs: competition with nucleotides and enhancement by translation initiation factor iso4G

Pokeweed antiviral protein (PAP) is a type I ribosomal inactivating protein (RIP). PAP binds to and depurinates the sarcin/ricin loop (SRL) of ribosomal RNA resulting in the cessation of protein synthesis. PAP has also been shown to bind to mRNA cap analogs and depurinate mRNA downstream of the cap structure. The biological role of cap binding and its possible role in PAP activity are not known. Here we show the first direct quantitative evidence for PAP binding to the cap analog m(7)GTP. We report a binding affinity of 43.3+/-0.1 nM at 25 degrees C as determined by fluorescence quenching experiments. This is similar to the values reported for wheat cap-binding proteins eIFiso4E and eIFiso4F. van't Hoff analysis of m(7)GTP-PAP equilibrium reveals a binding reaction that is enthalpy driven and entropy favored with TDeltaS degrees contributing 15% to the overall value of DeltaG degrees . This is in contrast to the wheat cap-binding proteins which are enthalpically driven in the DeltaG degrees for binding. Competition experiments indicate that ATP and GTP compete for the cap-binding site on PAP with slightly different affinities. Fluorescence studies of PAP-eIFiso4G binding reveal a protein-protein interaction with a K(d) of 108.4+/-0.3 nM. eIFiso4G was shown to enhance the interaction of PAP with m(7)GTP cap analog by 2.4-fold. These results suggest the involvement of PAP-translation initiation factor complexes in RNA selection and depurination.     

Poisson–Boltzmann model analysis of binding mRNA cap analogues to the translation initiation factor eIF4E

The electrostatic free energy of binding of two analogues of the 5′-mRNA cap, differing in size and electric charge, to the wild type and mutated eukaryotic initiation factor eIF4E was computed using the finite difference solutions to the Poisson–Boltzmann equation. Two definitions of the solute–solvent dielectric boundary were used: van der Waals model, solvent exclusion (SE) model. The computed electrostatic energies were supplemented by estimations of the non polar and entropic contributions. A comparison with experimental data for the investigated systems was done. It appears that the SE model with additional contribution fits experimental findings better than the van der Waals model does.     

The HRIGRXXR region of the DEAD box RNA helicase eukaryotic translation initiation factor 4A is required for RNA binding and ATP hydrolysis.

eIF-4A is a eukaryotic translation initiation factor that is required for mRNA binding to ribosomes. It exhibits single-stranded RNA-dependent ATPase activity, and in combination with a second initiation factor, eIF-4B, it exhibits duplex RNA helicase activity. eIF-4A is the prototype of a large family of proteins termed the DEAD box protein family, whose members share nine highly conserved amino acid regions. The functions of several of these conserved regions in eIF-4A have previously been assigned to ATP binding, ATPase, and helicase activities. To define the RNA-binding region of eIF-4A, a UV-induced cross-linking assay was used to analyze binding of mutant eIF-4A proteins to RNA. Mutants carrying mutations in the ATP-binding region (AXXXXGKT), ATPase region (DEAD), helicase region (SAT), and the most carboxy-terminal conserved region of the DEAD family, HRIGRXXR, were tested for RNA cross-linking. We show that mutations, either conservative or not, in any one of the three arginines in the HRIGRXXR sequence drastically reduced eIF-4A cross-linking to RNA. In addition, all the mutations in the HRIGRXXR region abrogate RNA helicase activity. Some but not all of these mutations affect ATP binding and ATPase activity. This is consistent with the hypothesis that the HRIGRXXR region is involved in the ATP hydrolysis reaction and would explain the coupling of ATPase and RNA-binding/helicase activities. Our results show that the HRIGRXXR region, which is QRXGRXXR or QXXGRXXR in the RNA and DNA helicases of the helicase superfamily II, is involved in ATP hydrolysis-dependent RNA interaction during unwinding. We also show that mutations in other regions of eIF-4A that abolish ATPase activity sharply decrease eIF-4A cross-linking to RNA. A model is proposed in which eIF-4A first binds ATP, resulting in a change in eIF-4A conformation which allows RNA binding that is dependent on the HRIGRXXR region. Binding of RNA induces ATP hydrolysis, leading to a more stable interaction with RNA. This process is then linked to unwinding of duplex RNA in the presence of eIF-4B.     

Positive heat capacity change upon specific binding of translation initiation factor eIF4E to mRNA 5' cap

Specific recognition of the mRNA 5' cap by eukaryotic initiation factor eIF4E is a rate-limiting step in the translation initiation. Fluorescence spectroscopy and high-sensitivity isothermal titration calorimetry were used to examine the thermodynamics of eIF4E binding to a cap-analogue, 7-methylGpppG. A van't Hoff plot revealed nonlinearity characterized by an unexpected, large positive molar heat capacity change (DeltaC(degree)(p) = +1.92 +/- 0.93 kJ.mol(-1).K(-1)), which was confirmed by direct ITC measurements (DeltaC(degree)(p) = +1.941 +/- 0.059 kJ.mol(-1).K(-1)). This unique result appears to come from an extensive additional hydration upon binding and charge-related interactions within the binding site. As a consequence of the positive DeltaC(degree)(p), the nature of the thermodynamic driving force changes with increasing temperature, from enthalpy-driven and entropy-opposed, through enthalpy- and entropy-driven in the range of biological temperatures, into entropy-driven and enthalpy-opposed. Comparison of the van't Hoff and calorimetric enthalpy values provided proof for the ligand protonation at N(1) upon binding, which is required for tight stabilization of the cap-eIF4E complex. Intramolecular self-stacking of the dinucleotide cap-analogue was analyzed to reveal the influence of this coupled process on the thermodynamic parameters of the eIF4E-mRNA 5' cap interaction. The temperature-dependent change in the conformation of 7-methylGpppG shifts significantly the intrinsic DeltaH(degree)(0) = -72.9 +/- 4.2 kJ.mol(-1) and DeltaS(degree)(0) = -116 +/- 58 J.mol(-1).K(-1) of binding to the less negative resultant values, by DeltaH(degree)(sst) = +9.76 +/- 1.15 kJ.mol(-1) and DeltaS(degree)(sst) = +24.8 +/- 2.1 J.mol(-1).K(-1) (at 293 K), while the corresponding DeltaC(degree)(p)(sst) = -0.0743 +/- 0.0083 kJ.mol(-1).K(-1) is negligible in comparison with the total DeltaC(degree)(p) .     

Physical association of eukaryotic initiation factor 4G (eIF4G) with eIF4A strongly enhances binding of eIF4G to the internal ribosomal entry site of encephalomyocarditis virus and is required for internal initiation of translation

Mammalian eukaryotic initiation factor 4GI (eIF4GI) may be divided into three similarly sized regions. The central region (amino acids [aa] 613 to 1090) binds eIF3, eIF4A, and the encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) and mediates initiation on this RNA. We identified the regions of eIF4GI that are responsible for its specific interaction with the IRES and that are required to mediate 48S complex formation on the IRES in vitro. Mutational analysis demarcated the IRES binding fragment of eIF4GI (aa 746 to 949) and indicated that it does not resemble an RNA recognition motif (RRM)-like domain. An additional amino-terminal sequence (aa 722 to 746) was required for binding eIF4A and for 48S complex formation. eIF4GI bound the EMCV IRES and beta-globin mRNA with similar affinities, but association with eIF4A increased its affinity for the EMCV IRES (but not beta-globin RNA) by 2 orders of magnitude. On the other hand, eIF4GI mutants with defects in binding eIF4A were defective in mediating 48S complex formation even if they bound the IRES normally. These data indicate that the eIF4G-eIF4A complex, rather than eIF4G alone, is required for specific high-affinity binding to the EMCV IRES and for internal ribosomal entry on this RNA.     

Binding of eukaryotic translation initiation factor 4E (eIF4E) to eIF4G represses translation of uncapped mRNA.

mRNA translation in crude extracts from the yeast Saccharomyces cerevisiae is stimulated by the cap structure and the poly(A) tail through the binding of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) and the poly(A) tail-binding protein Pab1p. These proteins also bind to the translation initiation factor eIF4G and thereby link the mRNA to the general translational apparatus. In contrast, uncapped, poly(A)-deficient mRNA is translated poorly in yeast extracts, in part because of the absence of eIF4E and Pab1p binding sites on the mRNA. Here, we report that uncapped-mRNA translation is also repressed in yeast extracts due to the binding of eIF4E to eIF4G. Specifically, we find that mutations which weaken the eIF4E binding site on the yeast eIF4G proteins Tif4631p and Tif4632p lead to temperature-sensitive growth in vivo and the stimulation of uncapped-mRNA translation in vitro. A mutation in eIF4E which disturbs its ability to interact with eIF4G also leads to a stimulation of uncapped-mRNA translation in vitro. Finally, overexpression of eIF4E in vivo or the addition of excess eIF4E in vitro reverses these effects of the mutations. These data support the hypothesis that the eIF4G protein can efficiently stimulate translation of exogenous uncapped mRNA in extracts but is prevented from doing so as a result of its association with eIF4E. They also suggest that some mRNAs may be translationally regulated in vivo in response to the amount of free eIF4G in the cell.