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Analysis of the mRNA Cap‐Binding Ability of Human Eukaryotic Initiation Factor‐4E by Use of Recombinant Wild‐Type and Mutant Forms
Abstract
In order to identify the amino acid residues necessary for the selective recognition of the mRNA cap structure by human eukaryotic initiation factor-4E (eIF-4E), which plays a central role in the first step of mRNA translation, we prepared recombinant wild-type and fourteen mutant forms and compared their cap-binding abilities by affinity chromatography. By the direct expression of a synthetic gene encoding human eIF-4E as the soluble form in Escherichia coli and the application on a 7-methylguanosine-5′-triphosphate–Sepharose 4B cap affinity column, pure recombinant eIF-4E was prepared; the optimum pH for the binding of the mRNA cap was 7.5. Among the amino acid residues conserved among various eIF-4E species, each of 14 functional residues was replaced with a nonpolar amino acid (alanine or leucine). All mutant eIF-4E genes, which were constructed by site-directed niutagenesis, were expressed in the same way as the wild type, and their cap-binding abilities were compared with that of the wild type. Consequently, all eight tryptophan residues, Glu103, and two histidine residues at positions 37 and 200 in human recombinant eIF-4E were suggested to be important for the recognition of the mRNA cap structure through direct interaction and/or indirect contributions. Indirect contributions included the construction of the overall protein structure, especially the cap-binding pocket.
... Evolutionary analyses were performed in MEGA7 [66]. [75]. In IbeIF4E, our results revealed a total of nine W residues that are identical amongst all species ( Figure 10). ...
... During purification we finally identified the 54- kDa polypeptide of bovine PARN after m 7 GTP- Sepharose chromatography (paper I). The m 7 GTP-Sepharose matrix partly resembles a methylated cap structure and has been used to purify cap-binding proteins (Morino et al., 1996). Similarly, the 74-kDa human, bovine and Xenopus PARN also bound to the m 7 GTP matrix (Copeland and Wormington, 2001; Gao et al., 2000; Körner et al., 1998). ...
Protein translation leading to polypeptide synthesis involves three distinct events namely initiation, elongation and termination. Translation initiation is a multi-step process that is carried out by ribosomes on the mRNA with the assistance of a large number of proteins called translation initiation factors. Trypanosomatids are kinetoplastidas (flagellated protozoans), some of which cause acute disease syndromes in humans. Vector-borne transmission of protozoan parasites like Leishmania and Trypanosoma causes diseases that affect a large section of the world population and lead to significant morbidity and mortality. The mechanisms of translation initiation in higher eukaryotes are relatively well understood. However, structural and functional conservation of initiation factors in trypanosomatids are only beginning to be understood. Studies carried out so far suggests that at least in Leishmania and Trypanosoma eIF4E function may not be restricted to canonical translation initiation and some of the homologues may have alternate/non-canonical functions. Nonetheless, all of them bind the cap analogues, albeit with different efficiencies, indicating that this property may play an important role in the functionality of eIF4Es. Here, I give a brief background of trypanosomatid eIF4Es and revisit the cap-binding signatures of eIF4E orthologues in trypanosomatids, whose genome sequences are available, in detail, in comparison to human eIF4E1 and Trypanosoma cruzi eIF4E5, with an expanded list of members of this group in light of newer findings. The group 1 and 2 eIF4Es may use either a variation of heIF4E1 or T. cruzi eIF4E5 cap-4-binding signatures while eIF4E5 and eIF4E6 use distinct amino acid contacts.
P311, a conserved 8-kDa intracellular protein expressed in brain, smooth muscle, regenerating tissues, and malignant glioblastomas,
represents the first documented stimulator of TGF-β1-3 translation in vitro and in vivo. Here we initiated efforts to define the mechanism underlying P311 function. PONDR® (Predictor Of Naturally Disordered Regions)
analysis suggested and CD confirmed that P311 is an intrinsically disordered protein, therefore requiring an interacting partner
to acquire tertiary structure and function. Immunoprecipitation coupled with mass spectroscopy identified eIF3 subunit b (eIF3b)
as a novel P311 binding partner. Immunohistochemical colocalization, GST pulldown, and surface plasmon resonance studies revealed
that P311-eIF3b interaction is direct and has a Kd of 1.26 μm. Binding sites were mapped to the non-canonical RNA recognition motif of eIF3b and a central 11-amino acid-long region of
P311, here referred to as eIF3b binding motif. Disruption of P311-eIF3b binding inhibited translation of TGF-β1, 2, and 3,
as indicated by luciferase reporter assays, polysome fractionation studies, and Western blot analysis. RNA precipitation assays
after UV cross-linking and RNA-protein EMSA demonstrated that P311 binds directly to TGF-β 5′UTRs mRNAs through a previously
unidentified RNA recognition motif-like motif. Our results demonstrate that P311 is a novel RNA-binding protein that, by interacting
with TGF-βs 5′UTRs and eIF3b, stimulates the translation of TGF-β1, 2, and 3.
抄録
Information on the conformational feature and specific intermolecular interaction of biomolecules is important to understand the biological function and to develop device for treating disorder caused by the abnormal function. Thus the 3D structures of the biologically active molecules and the specific interactions with their target molecules at the atomic level have been investigated by various physicochemical approaches. Herein, the following five subjects are reviewed: (1) function-linked conformations of biomolecules including natural annular products, opioid peptides and neuropeptides; (2) π-π stacking interactions of tryptophan derivatives with coenzymes and nucleic acid bases; (3) mRNA cap recognition of eukaryotic initiation factor 4E and its regulation by 4E-binding protein; (4) conformational feature of histamine H2 receptor antagonists and design of cathepsin B inhibitors; (5) self-aggregation mechanism of tau protein and its inhibition.
This article summarizes methods for increasing the fraction of recombinant proteins expressed in a soluble form in the cytoplasmic fraction of bacteria. Many recombinant proteins, when expressed at very high levels in bacteria, form insoluble aggregates called inclusion bodies (IBs). Growth conditions can be manipulated to keep protein in a soluble form, including lowering the temperature during induction, choice of host strain and expression vector, or coculturing of proteins that assist in folding. Higher yields can be obtained by mutating the protein or fusing it with various linkers (which can also improve purification by enhancing column chromatography interactions). Eliminating protease or RNase recognition sites can stabilize the protein or mRNA, as can mutations that improve recognition by cofactors that assist in folding.
Methodology to simultaneously produce many different recombinant proteins in a soluble form has been developed as part of the structural genomics initiative; these can also be used to for scanning mutagenesis studies. High density bacterial cultivation can be used to produce gram per liter quantities of recombinant proteins at the industrial scale. However, maintaining high levels of soluble protein requires optimizing all aspects of growth, ranging from how the inoculum is stored, to medium details and bacterial harvesting and lysis.
The eukaryotic initiation factor 4E (eIF4E) serves as a master switch that controls mRNA translation through the promotive binding to eIF4G and the regulative binding with the endogenous inhibitor 4E-BP. Although the bindings of eIF4G and 4E-BP to eIF4E proceed through the common eIF4E recognition Y(X)(4)Lφ motif (X: variable, φ: hydrophobic) (first binding site), the relationship between their eIF4E binding mode and the functional difference is hardly known. Recently, we have clarified the existence and function of the second eIF4E binding site in 4E-BP. Surface plasmon resonance (SPR) analysis based on the sequential comparison between 4E-BP and eIF4GI clarified that eIF4G has the second binding site at the periphery of the (597)SDVVL(601) sequence and that it plays an auxiliary but indispensable function in stabilizing the binding of the first binding sequence (572)YDREFLL(578). The kinetic parameters of the interactions of the eIF4GI and 4E-BP2 fragment peptides with eIF4E showed that the association (ka) and dissociation (kd) rates of the former peptide are about three and two orders of magnitude lower than those of the latter peptide, respectively. This means that eIF4G has a potent resistive property for release from eIF4E, although its rate of binding to eIF4E is not as high as that of 4E-BP, that is, 4E-BP is apt to bind to and be released from eIF4E, as compared with eIF4G. Isothermal titration calorimetry (ITC) showed the opposite behavior between the second binding sites of eIF4GI and 4E-BP for the interaction with eIF4E. This clearly indicates the importance of the second binding region for the difference in function between eIF4G and 4E-BP for eIF4E translation.
Although the central α-helical Y(X)4LΦ motif (X, variable amino acid; Φ, hydrophobic amino acid) of the translational regulator 4E-BP [eIF (eukaryotic initiation factor) 4E-binding protein] is the core binding region for the mRNA cap-binding protein eIF4E, the functions of its N- and C-terminal flexible regions for interaction with eIF4E remain to be elucidated. To identify the role for the C-terminal region in such an interaction, the binding features of full-length and sequential C-terminal deletion mutants of 4E-BPn (n=1-3) subtypes were investigated by SPR (surface plasmon resonance) analysis and ITC (isothermal titration calorimetry). Consequently, the conserved PGVTS/T motif within the C-terminal region was shown to act as the second binding region and to play an important role in the tight binding to eIF4E. The 4E-BP subtypes increased the association constant with eIF4E by approximately 1000-fold in the presence of this conserved region compared with that in the absence of this region. The sequential deletion of this conserved region in 4E-BP1 showed that deletion of Val81 leads to a considerable decrease in the binding ability of 4E-BP. Molecular dynamics simulation suggested that the conserved PGVTS/T region functions as a kind of paste, adhering the root of both the eIF4E N-terminal and 4E-BP C-terminal flexible regions through a hydrophobic interaction, where valine is located at the crossing position of both flexible regions. It is concluded that the conserved PGVTS/T motif within the flexible C-terminus of 4E-BP plays an auxiliary, but indispensable, role in strengthening the binding of eIF4E to the core Y(X)4LΦ motif.
To clarify the higher eukaryotic initiation factor 4E (eIF4E) binding selectivity of 4E-binding protein 2 (4E-BP2) than of 4E-BP1, as determined by Trp fluorescence analysis, the crystal structure of the eIF4E binding region of 4E-BP2 in complex with m(7) GTP-bound human eIF4E has been determined by X-ray diffraction analysis and compared with that of 4E-BP1. The crystal structure revealed that the Pro47-Ser65 moiety of 4E-BP2 adopts a L-shaped conformation involving extended and α-helical structures and extends over the N-terminal loop and two different helix regions of eIF4E through hydrogen bonds, and electrostatic and hydrophobic interactions; these features were similarly observed for 4E-BP1. Although the pattern of the overall interaction of 4E-BP2 with eIF4E was similar to that of 4E-BP1, a notable difference was observed for the 60-63 sequence in relation to the conformation and binding selectivity of the 4E-BP isoform, i.e. Met-Glu-Cys-Arg for 4E-BP1 and Leu-Asp-Arg-Arg for 4E-BP2. In this paper, we report that the structural scaffold of the eIF4E binding preference for 4E-BP2 over 4E-BP1 is based on the stacking of the Arg63 planar side chain on the Trp73 indole ring of eIF4E and the construction of a compact hydrophobic space around the Trp73 indole ring by the Leu59-Leu60 sequence of 4E-BP2.
Information on the structural basis of intermolecular recognition or self-aggregation of biomolecules at the atomic level is important to understand biological functions and to develop devices for treating disorders caused by abnormal functions. Thus structural analysis of specific intermolecular or intramolecular interactions of biomolecules has been performed using various physicochemical approaches. Herein, the following three subjects are reviewed: (1) structural analyses of mRNA cap structure recognition by eukaryotic initiation factor 4E and its functional regulation by endogenous 4E-binding protein; (2) structural studies of self-aggregation mechanism of microtubule-binding domain in tau protein and aggregation inhibitor; and (3) molecular design of cathepsin B-specific inhibitor.
The specific recognition by proteins of the 5' and 3' ends of RNA molecules is an important facet of many cellular processes, including RNA maturation, regulation of translation initiation and control of gene expression by degradation and RNA interference. The aim of this review is to survey recent structural analyses of protein binding domains that specifically bind to the extreme 5' or 3' termini of RNA. For reasons of space and because their interactions are also governed by catalytic considerations, we have excluded enzymes that modify the 5' and 3' extremities of RNA. It is clear that there is enormous structural diversity among the proteins that have evolved to bind to the ends of RNA molecules. Moreover, they commonly exhibit conformational flexibility that appears to be important for binding and regulation of the interaction. This flexibility has sometimes complicated the interpretation of structural results and presents significant challenges for future investigations.
RNAs of many positive strand RNA viruses lack a 5′ cap structure and instead rely on cap-independent translation elements
(CITEs) to facilitate efficient translation initiation. The mechanisms by which these RNAs recruit ribosomes are poorly understood,
and for many viruses the CITE is unknown. Here we identify the first CITE of an umbravirus in the 3′-untranslated region of
pea enation mosaic virus RNA 2. Chemical and enzymatic probing of the ∼100-nucleotide PEMV RNA 2 CITE (PTE), and mutagenesis revealed that it forms a long, bulged helix that branches into two short stem-loops, with a possible
pseudoknot interaction between a C-rich bulge at the branch point and a G-rich bulge in the main helix. The PTE inhibited
translation in trans, and addition of eIF4F, but not eIFiso4F, restored translation. Filter binding assays revealed that the PTE binds eIF4F and
its eIF4E subunit with high affinity. Tight binding required an intact cap-binding pocket in eIF4E. Among many PTE mutants,
there was a strong correlation between PTE-eIF4E binding affinity and ability to stimulate cap-independent translation. We
conclude that the PTE recruits eIF4F by binding eIF4E. The PTE represents a different class of translation enhancer element,
as defined by its structure and ability to bind eIF4E in the absence of an m7G cap.
To investigate the binding specificity of turnip mosaic virus (TuMV) viral protein-genome linked (VPg) with translation initiation factor 4E, we evaluated here the kinetic parameters for the interactions of human eIF4E, Caenorhabditis elegans IFE-3 and IFE-5 and Arabidopsis eIFiso4E, by surface plasmon resonance (SPR). The results indicated that TuMV VPg does not show a binding preference for Arabidopsis eIFiso4E, even though it is from a host species whereas the other eIF4E orthologues are not. Surprisingly, the effect of m(7)GTP on both the rate constants and equilibrium binding constants for the interactions of VPg differed for the four eIF4E orthologues. In the case of eIFiso4E and IFE-3, m(7)GTP increased k(on), but for eIF4E and IFE-5, it decreased k(on). To provide insight into the structural basis for these differences in VPg binding, tertiary structures of the eIF4E orthologues were predicted on the basis of the previously determined crystal structure of m(7)GpppA-bound human eIF4E. The results suggested that in cap-bound eIF4E orthologues, the VPg binds to the C-terminal region, which constitutes one side of the entrance to the cap-binding pocket, whereas in the cap-free state, VPg binds to the widely opened cap-binding pocket and its surrounding region. The binding of VPg to the C-terminal region was confirmed by the SPR analyses of N- or C-terminal residues-deleted eIF4E orthologues.
Although the alpha-helical Y(X)4Lvarphi containing region of eIF4E-binding protein (4EBP) is the major binding region with eukaryotic initiation factor 4E (eIF4E), the roles of its N- and C-terminal regions in the binding are hardly known. To clarify the roles of these flexible regions in the interaction, the binding features of the sequentially N-, C-, or both-terminal-residue-deleted 4EBP2 mutants were investigated by surface plasmon resonance (SPR) analysis. It was shown that the C-terminal His74-Glu89 sequence has an auxiliary, but indispensable, function in stabilizing the binding to eIF4E. The possible interaction with eIF4E was estimated by molecular dynamics simulation. This is the first report on the importance of the C-terminal flexible region in the eIF4E-binding regulation of 4EBP.
Proteins interacting with messenger RNAs (mRNAs) affect their nuclear processing, export, translation efficiency, stability, or cytoplasmic localization. Such RNA-binding proteins are often modular, containing RNA-binding domain(s) and other functional modules. To analyze the function of such proteins independent of their normal RNA-binding domains or to introduce effector modules to defined RNA-binding regions, a number of tethering approaches have been developed, often based on the use of large proteins and their specifically interacting RNA sequences. Here we report the use of a versatile system to tether proteins to mRNAs. The 22 amino acid RNA-binding domain of the lambda bacteriophage antiterminator protein N (lambdaN-(1-22) or lambdaN peptide) is used to tag the protein of interest, and its specific 19 nt binding site (boxB) is inserted into the target RNA recruiting the properties of the fusion protein to the RNA. The major advantage of this system derives from the small size of the peptide and its target sequence, which facilitates cloning and its use for biochemical experiments and diminishes possible interferences with the fused protein. The chapter illustrates the use of this system to create dedicated mRNA-specific factors involved in processes, such as mRNA translation and nonsense-mediated mRNA decay.
The translational factor eukaryotic initiation factor 4E (eIF4E) is a central component in the initiation and regulation of translation in eukaryotic cells. Through its interaction with the 5' cap structure of mRNA, eIF4E functions to recruit mRNAs to the ribosome. The accumulation of expressed sequence tag sequences has allowed the identification of three different eIF4E-family members in mammals termed eIF4E-1, eIF4E-2 (4EHP, 4E-LP) and eIF4E-3, which differ in their structural signatures, functional characteristics and expression patterns. Unlike eIF4E-1, which is found in all eukaryotes, orthologues for eIF4E-2 appear to be restricted to metazoans, while those for eIF4E-3 have been found only in chordates. Like prototypical eIF4E-1, eIF4E-2 was found to be ubiquitously expressed, with the highest levels in the testis. Expression of eIF4E-3 was detected only in heart, skeletal muscle, lung and spleen. Similarly to eIF4E-1, both eIF4E-2 and eIF4E-3 can bind to the mRNA cap-structure. However, in contrast to eIF4E-1 which interacts with both the scaffold protein, eIF4G and the translational repressor proteins, the eIF4E-binding proteins (4E-BPs), eIF4E-2 and eIF4E-3 each possesses a range of partial activities. eIF4E-2 does not interact with eIF4G, but does interact with 4E-BPs. Conversely, eIF4E-3 interacts with eIF4G, but not with 4E-BPs. Neither eIF4E-2 nor eIF4E-3 is able to rescue the lethality of eIF4E gene deletion in yeast. It is hypothesized that each eIF4E-family member fills a specialized niche in the recruitment of mRNAs by the ribosome through differences in their abilities to bind cap and/or to interact with eIF4G and the 4E-BPs.
From the characterization of the recessive resistance gene, sbm1, in pea we have identified the eukaryotic translation initiation factor, eIF4E, as a susceptibility factor required for infection with the Potyvirus, Pea seed-borne mosaic virus. A functional analysis of the mode of action of the product of the dominant allele revealed a novel function for eIF4E in its support for virus movement from cell-to-cell, in addition to its probable support for viral RNA translation, and hence replication. Different resistance specificities in two independent pea lines were explained by different mutations in eIF4E. On the modelled structure of eIF4E the coding changes were in both cases lying in and around the structural pocket involved in binding the 5'-m7G cap of eukaryotic mRNAs. Protein expression and cap-binding analysis showed that eIF4E encoded by a resistant plant could not bind to m7G-Sepharose, a result which may point to functional redundancy between eIF4E and the paralogous eIF(iso)4E in resistant peas. These observations, together with related findings for other potyvirus recessive resistances, provide a more complete picture of the potyvirus life cycle.
The eukaryotic translation initiation factor 4F (eIF4F) consists of three polypeptides (eIF4A, eIF4G, and eIF4E) and is responsible for recruiting ribosomes to mRNA. eIF4E recognizes the mRNA 5'-cap structure (m7GpppN) and plays a pivotal role in control of translation initiation, which is the rate-limiting step in translation. Overexpression of eIF4E has a dramatic effect on cell growth and leads to oncogenic transformation. Therefore, an inhibitory agent to eIF4E, if any, might serve as a novel therapeutic against malignancies that are caused by aberrant translational control. Along these lines, we developed two RNA aptamers, aptamer 1 and aptamer 2, with high affinity for mammalian eIF4E by in vitro RNA selection-amplification. Aptamer 1 inhibits the cap binding to eIF4E more efficiently than the cap analog m7GpppN or aptamer 2. Consistently, aptamer 1 inhibits specifically cap-dependent in vitro translation while it does not inhibit cap-independent HCV IRES-directed translation initiation. The interaction between eIF4E and eIF4E-binding protein 1 (4E-BP1), however, was not inhibited by aptamer 1. Aptamer 1 is composed of 86 nucleotides, and the high affinity to eIF4E is affected by deletions at both termini. Moreover, relatively large areas in the aptamer 1 fold are protected by eIF4E as determined by ribonuclease footprinting. These findings indicate that aptamers can achieve high affinity to a specific target protein via global conformational recognition. The genetic mutation and affinity study of variant eIF4E proteins suggests that aptamer 1 binds to eIF4E adjacent to the entrance of the cap-binding slot and blocks the cap-binding pocket, thereby inhibiting translation initiation.
Most cellular and eukaryotic viral mRNAs have a cap structure at their 5' end that is critical for efficient translation. Cap structures also aid in mRNA transport from nucleus to cytoplasm and, in addition, protect the mRNAs from degradation by 5' exonucleases. Cap function is mediated by cap-binding proteins that play a key role in translational control. Recent structural studies on the cellular cap-binding complex, the eukaryotic translation initiation factor 4E and the vaccinia virus protein 39, suggest that these three evolutionary unrelated cap-binding proteins have evolved a common cap-binding pocket by convergent evolution. In this pocket the positively charged N(7)-methylated guanine ring of the cap structure is stacked between two aromatic amino acids. In this review, the similarities and differences in cap binding by these three different cap-binding proteins are discussed. A comparison with new functional data for another viral cap-binding protein--the polymerase basic protein (PB2) of influenza virus--suggests that a similar cap-binding mechanism has also evolved in influenza virus.
Eukaryotic translation initiation factor 4E (eIF4E) binds to the m7GTP of capped mRNAs and is an essential component of the translational machinery that recruits the 40S small ribosomal subunit.
We describe here the identification and characterization of two eIF4E homologues in an ancient protist, Giardia lamblia. Using m7GTP-Sepharose affinity column chromatography, a specific binding protein was isolated and identified as Giardia eIF4E2. The other homologue, Giardia eIF4E1, bound only to the m2,2,7GpppN structure. Although neither homologue can rescue the function of yeast eIF4E, a knockdown of eIF4E2 mRNA in Giardia by a virus-based antisense ribozyme decreased translation, which was shown to use m7GpppN-capped mRNA as a template. Thus, eIF4E2 is likely the cap-binding protein in a translation initiation complex. The same
knockdown approach indicated that eIF4E1 is not required for translation in Giardia. Immunofluorescence assays showed wide distribution of both homologues in the cytoplasm. But eIF4E1 was also found concentrated
and colocalized with the m2,2,7GpppN cap, 16S-like rRNA, and fibrillarin in the nucleolus-like structure in the nucleus. eIF4E1 depletion from Giardia did not affect mRNA splicing, but the protein was bound to Giardia small nuclear RNAs D and H known to have an m2,2,7GpppN cap, thus suggesting a novel function not yet observed among other eIF4Es in eukaryotes.
Taking advantage of the Trp73 residue located close to the 4E-BP binding site of eIF4E, the interaction between the 4E-BP isoform and eIF4E was investigated by the Trp fluorescence titration method. Although no significant difference was observed among the association constants of three 4E-BP isoforms, the binding preference of 4E-BP2 over 4E-BP1 and -BP3 was shown, probably due to the effect of a 4E-BP2-specific LDRR (60-63) sequence for the binding with eIF4E. By contrast, surface plasmon resonance (SPR) analyses showed the binding preference of 4E-BP1, although the difference among the isoforms was also not significant. This inconsistency with fluorescence analysis likely resulted from the different observation points of the interaction, i.e., local and overall interactions observed by the fluorescence and SPR methods, respectively. To clarify the structural basis for these spectroscopic results, the crystal structure of the ternary complex of m7GpppA-eIF4E-4E-BP1 fragment (Thr36-Thr70) was analyzed by the X-ray diffraction method. Crystal structure analysis at 2.1 A resolution revealed that the 4E-BP1 fragment, assigned to the Pro47-Pro66 peptide moiety, adopted a reverse L-shaped conformation involving the beta sheet and alpha-helical structures and was located at the root of the handle of the temple-bell-shaped eIF4E through hydrophilic and hydrophobic interactions. Based on the observed binding mode, possible interactions with the three 4E-BP isoforms have been discussed. On the other hand, since the crystal structural comparison with the previously determined m7GpppA-eIF4E-4E binary complex showed that the docking of the 4E-BP1 fragment does not significantly affect the overall tertiary structure and cap-binding scaffold of eIF4E, the dynamic regulation of the cap-binding of eIF4E by 4E-BP1 was investigated by molecular dynamics (MD) simulations. Consequently, the simulation suggested that (i) the helical region of the 4E-BP1 peptide is important for the binding with eIF4E, (ii) the existence of a cap structure stabilizes the binding of eIF4E with 4E-BP, (iii) the binding of 4E-BP stabilizes the cap-binding pocket of eIF4E, and (iv) the phosphorylation of Ser67 alone does not induce the separation of 4E-BP from eIF4E, but increases the structural rigidity of 4E-BP. These results provide the structural basis for the mRNA cap-binding regulation of eIF4E by 4E-BP.
To clarify the contribution of N-terminal region of eukaryotic initiation factor 4E (eIF4E) to the interaction with 4E-BP and to investigate the effect of 4E-BP phosphorylation on the interaction with eIF4E, the interaction profiles of the Ser65-unphosphorylated and phosphorylated peptides (Thr37-Thr70 fragment of 4E-BP1) with full-length and N-terminal 33 residues-deleted eIF4Es were investigated by fluorescence and SPR methods. The effect of N-terminal region of eIF4E on the interaction with 4E-BP1 peptides was shown to be dependent on the interaction state, that is, the steady-state fluorescence and kinetic-state SRP analyses showed the positive and negative contributions of the N-terminal region to the interaction with the peptide, respectively, despite its unphosphorylated or phosphorylated state. The comparison of the association constants of the peptide with those of full-length 4E-BP1 indicated the importance of N-terminal (1-36) and/or C-terminal (71-118) sequence of 4E-BP1 for the interaction, although the MD simulations suggested that the alpha-helical region (Arg56-Cys62) of 4E-BP1 peptide is sufficient for keeping the interaction. The MD simulations also indicated that a charge-dependent rigid hydration shell formed around the phosphate group makes the molecular conformation rigid, and single Ser65 phosphorylation is insufficient for releasing 4E-PB1 peptide from eIF4E.
Kinetoplastid mRNAs possess a unique hypermethylated cap 4 structure derived from the standard m7GpppN cap structure, with 2′-O methylations on the first four ribose sugars and additional base methylations on the first
adenine and the fourth uracil. While the enzymes responsible for m7GpppN cap 0 formations has been characterized in Trypanosoma brucei, the mechanism of cap 4 methylation and the role of the hypermethylated structure remain unclear. Here, we describe the characterization
of a 48 kDa T.brucei 2′-O nucleoside methyltransferase (TbCom1). Recombinant TbCom1 transfers the methyl group from S-adenosylmethionine (AdoMet)
to the 2′-OH of the second nucleoside of m7GpppNpNp-RNA to form m7GpppNpNmp-RNA. TbCom1 is also capable of converting cap 1 RNA to cap 2 RNA. The methyl transfer reaction is dependent on the
m7GpppN cap, as the enzyme does not form a stable interaction with GpppN-terminated RNA. Mutational analysis establishes that
the TbCom1 and vaccinia virus VP39 methyltransferases share mechanistic similarities in AdoMet- and cap-recognition. Two aromatic
residues, Tyr18 and Tyr187, may participate in base-stacking interactions with the guanine ring of the cap, as the removal
of each of these aromatic side-chains abolishes cap-specific RNA-binding.
The 5′ cap structure of trypanosomatid mRNAs, denoted cap 4, is a complex structure that contains unusual modifications on
the first four nucleotides. We examined the four eukaryotic initiation factor 4E (eIF4E) homologues found in the Leishmania genome database. These proteins, denoted LeishIF4E-1 to LeishIF4E-4, are located in the cytoplasm. They show only a limited
degree of sequence homology with known eIF4E isoforms and among themselves. However, computerized structure prediction suggests
that the cap-binding pocket is conserved in each of the homologues, as confirmed by binding assays to m7GTP, cap 4, and its intermediates. LeishIF4E-1 and LeishIF4E-4 each bind m7GTP and cap 4 comparably well, and only these two proteins could interact with the mammalian eIF4E binding protein 4EBP1,
though with different efficiencies. 4EBP1 is a translation repressor that competes with eIF4G for the same residues on eIF4E;
thus, LeishIF4E-1 and LeishIF4E-4 are reasonable candidates for serving as translation factors. LeishIF4E-1 is more abundant
in amastigotes and also contains a typical 3′ untranslated region element that is found in amastigote-specific genes. LeishIF4E-2
bound mainly to cap 4 and comigrated with polysomal fractions on sucrose gradients. Since the consensus eIF4E is usually found
in 48S complexes, LeishIF4E-2 could possibly be associated with the stabilization of trypanosomatid polysomes. LeishIF4E-3
bound mainly m7GTP, excluding its involvement in the translation of cap 4-protected mRNAs. It comigrates with 80S complexes which are resistant
to micrococcal nuclease, but its function is yet unknown. None of the isoforms can functionally complement the Saccharomyces cerevisiae eIF4E, indicating that despite their structural conservation, they are considerably diverged.
To investigate the binding preference of eIF4E for the three eIF4E-binding isoforms (4E-BP1-3) and the function of N-terminal flexible region of eIF4E for their interactions, the binding parameters of recombinant full-length and N-terminal residues-deleted eIF4Es with 4E-BP1-3 were investigated by the surface plasmon resonance (SPR) analysis. Consequently, it was clarified that 4E-BP2 exhibits the highest binding affinity for both m7GTP-bound and -unbound full-length eIF4Es when compared with 4E-BP1 and 4E-BP3. This is primarily due to the difference among their dissociation rates, because their association rates are almost the same. Interestingly, the deletion of the 33 N-terminal residues of eIF4E increased its binding affinities for 4E-BP1 and 4E-BP2 markedly, whereas such a change was not observed by at least the N-terminal deletion up to 26 residues. In contrast, the binding parameters of 4E-BP3 were hardly influenced by N-terminal deletion up to 33 residues. From the comparison of the amino acid sequences of 4E-BP1-3, the present result indicates the importance of N-terminal flexible region of eIF4E for the suppressive binding with 4E-BP1 and 2, together with the possible contribution of N-terminal sequence of 4E-BP isoform to the regulative binding to eIF4E.
Ribosome recruitment to the majority of eukaryotic mRNAs is facilitated by the interaction of the cap binding protein, eIF4E, with the mRNA 5' cap structure. eIF4E stimulates translation through its interaction with a scaffolding protein, eIF4G, which helps to recruit the ribosome. Metazoans also contain a homolog of eIF4E, termed 4EHP, which binds the cap structure, but not eIF4G, and thus cannot stimulate translation, but it instead inhibits the translation of only one known, and possibly subset mRNAs. To understand why 4EHP does not inhibit general translation, we studied the binding affinity of 4EHP for cap analogs using two methods: fluorescence titration and stopped-flow measurements. We show that 4EHP binds cap analogs m(7)GpppG and m(7)GTP with 30 and 100 lower affinity than eIF4E. Thus, 4EHP cannot compete with eIF4E for binding to the cap structure of most mRNAs.
Mimivirus isolated from A. polyphaga is the largest virus discovered so far. It is unique among all the viruses in having genes related to translation, DNA repair and replication which bear close homology to eukaryotic genes. Nevertheless, only a small fraction of the proteins (33%) encoded in this genome has been assigned a function. Furthermore, a large fraction of the unassigned protein sequences bear no sequence similarity to proteins from other genomes. These sequences are referred to as ORFans. Because of their lack of sequence similarity to other proteins, they can not be assigned putative functions using standard sequence comparison methods. As part of our genome-wide computational efforts aimed at characterizing Mimivirus ORFans, we have applied fold-recognition methods to predict the structure of these ORFans and further functions were derived based on conservation of functionally important residues in sequence-template alignments.
Using fold recognition, we have identified highly confident computational 3D structural assignments for 21 Mimivirus ORFans. In addition, highly confident functional predictions for 6 of these ORFans were derived by analyzing the conservation of functional motifs between the predicted structures and proteins of known function. This analysis allowed us to classify these 6 previously unannotated ORFans into their specific protein families: carboxylesterase/thioesterase, metal-dependent deacetylase, P-loop kinases, 3-methyladenine DNA glycosylase, BTB domain and eukaryotic translation initiation factor eIF4E.
Using stringent fold recognition criteria we have assigned three-dimensional structures for 21 of the ORFans encoded in the Mimivirus genome. Further, based on the 3D models and an analysis of the conservation of functionally important residues and motifs, we were able to derive functional attributes for 6 of the ORFans. Our computational identification of important functional sites in these ORFans can be the basis for a subsequent experimental verification of our predictions. Further computational and experimental studies are required to elucidate the 3D structures and functions of the remaining Mimivirus ORFans.
Naturally existing variation in the eukaryotic translation initiation factor 4E (eIF4E) homolog encoded at the pvr1 locus in Capsicum results in recessively inherited resistance against several potyviruses. Previously reported data indicate that the physical interaction between Capsicum-eIF4E and the viral genome-linked protein (VPg) is required for the viral infection in the Capsicum-Tobacco etch virus (TEV) pathosystem. In this study, the potential structural role(s) of natural variation in the eIF4E protein encoded by recessive resistance alleles and their biological consequences have been assessed. Using high-resolution three-dimensional structural models based on the available crystallographic structures of eIF4E, we show that the amino acid substitution G107R, found in many recessive plant virus resistance genes encoding eIF4E, is predicted to result in a substantial modification in the protein binding pocket. The G107R change was shown to not only be responsible for the interruption of VPg binding in planta but also for the loss of cap binding ability in vitro, the principal function of eIF4E in the host. Overexpression of the Capsicum-eIF4E protein containing the G107R amino acid substitution in Solanum lycopersicum indicated that this polymorphism alone is sufficient for the acquisition of resistance against several TEV strains.
Eukaryotic translation initiation factor 4E (eIF4E) is an essential component of the translational machinery that binds m(7)GTP and mediates the recruitment of capped mRNAs by the small ribosomal subunit. Recently, a number of proteins with homology to eIF4E have been reported in plants, invertebrates, and mammals. Together with the prototypical translation factor, these constitute a new family of structurally related proteins. To distinguish the prototypical translation factor eIF4E from other family members, it has been termed eIF4E-1 (Keiper, B. D., Lamphear, B. J., Deshpande, A. M., Jankowska-Anyszka, M., Aamodt, E. J., Blumenthal, T., and Rhoads, R. E. (2000) J. Biol. Chem. 275, 10590-10596). We describe the characterization of two eIF4E family members in the zebrafish Danio rerio. Based on their relative identities with human eIF4E-1, these zebrafish proteins are termed eIF4E-1A (82%) and eIF4E-1B (66%). eIF4E-1B, originally termed eIF4E(L), has been reported previously as the zebrafish eIF4E-1 counterpart (Fahrenkrug, S. C., Dahlquist, M. O., Clark, K., and Hackett, P. B. (1999) Differentiation 65, 191-201; Fahrenkrug, S. C., Joshi, B., Hackett, P. B., and Jagus, R. (2000) Differentiation 66, 15-22). Sequence comparisons suggest that the two genes probably evolved from a duplication event that occurred during vertebrate evolution. eIF4E-1A is expressed ubiquitously in zebrafish, whereas expression of eIF4E-1B is restricted to early embryonic development and to gonads and muscle of the tissues investigated. The ability of these two zebrafish proteins to bind m(7)GTP, eIF4G, and 4E-BP, as well as to complement yeast conditionally deficient in functional eIF4E, show that eIF4E-1A is a functional equivalent of human eIF4E-1. Surprisingly, although eIF4E-1B possesses all known residues thought to be required for interaction with the cap structure, eIF4G, and 4E-BPs, it fails to interact with any of these components, suggesting that this protein serves a role other than that assigned to eIF4E.
The eukaryotic translation initiation factor 4E (eIF4E) (the cap-binding protein) is involved in natural resistance against
several potyviruses in plants. In lettuce, the recessive resistance genes mo11 and mo12 against Lettuce mosaic virus (LMV) are alleles coding for forms of eIF4E unable, or less effective, to support virus accumulation. A recombinant LMV expressing
the eIF4E of a susceptible lettuce variety from its genome was able to produce symptoms in mo11 or mo12 varieties. In order to identify the eIF4E amino acid residues necessary for viral infection, we constructed recombinant LMV
expressing eIF4E with point mutations affecting various amino acids and compared the abilities of these eIF4E mutants to complement
LMV infection in resistant plants. Three types of mutations were produced in order to affect different biochemical functions
of eIF4E: cap binding, eIF4G binding, and putative interaction with other virus or host proteins. Several mutations severely
reduced the ability of eIF4E to complement LMV accumulation in a resistant host and impeded essential eIF4E functions in yeast.
However, the ability of eIF4E to bind a cap analogue or to fully interact with eIF4G appeared unlinked to LMV infection. In
addition to providing a functional mutational map of a plant eIF4E, this suggests that the role of eIF4E in the LMV cycle
might be distinct from its physiological function in cellular mRNA translation.
Production of recombinant proteins in bacteria is limited by the formation of cytoplasmic aggregates (inclusion bodies or "IBs"). This review summarizes what is known about why IBs form and ways of increasing the production of soluble protein in bacterial systems. The easiest way to lower IB formation is to reduce the growth temperature of the bacteria. IB formation is not directly correlatable with the production rate, nor with the size of the produced protein. The primary sequences of a few proteins that do not form IBs at higher production temperatures contain either a low content of proline residues or stretches of acidic amino acids. Metal ion binding may also lower the tendency to form IBs at growth temperatures above 30°C. Three aspects of protein synthesis in mammalian cells, compartmentation, interprotein interactions (sortases, foldases, unfoldases, and chaperonins), and post-translational modifications, have significant effects on the solubility of the proteins produced. Possibilities for mimicking these mechanisms in bacteria via secretion, cloning of mammalian foldases, and mutation of the post-translational modification systems of the host bacteria are discussed.
The 5' cap structure of eucaryotic mRNA plays a pivotal role in mRNA metabolism. This report demonstrates that anti-sense oligonucleotides equipped with 3'-overhanging nucleotides modulate the amount of recombinant human eucaryotic initiation factor-4E that binds to a 5'-capped oligoribonucleotide. The degree of inhibition or enhancement of protein binding is dependent upon the number and sequence of overhanging nucleotides. A 45% inhibition of complexation was observed by the addition of one 3'-overhanging guanosine residue. Addition of a second residue (+2/GN) resulted in a higher degree of inhibition, 77-88%. In contrast, addition of one adenosine residue enhanced the formation of the eucaryotic initiation factor-4E-m7GpppRNA complex by 213%. Modulation of protein interactions with the 5'-cap structure is likely to effect several biological events, including pre-mRNA processing, transport of the mRNA from the nucleus to the cytoplasm and translation of the target mRNA. This targeting strategy in anti-sense chemistry may have practical applications in experimental biology and medicine.
We have identified a cap binding protein in a HeLa nuclear extract using a gel mobility shift assay probed with capped RNA.
Subcellular fractionation of HeLa cells revealed that the majority (about 70%) of the cap binding activity is present in the
nuclear extract, about 20% is in the cytoplasmic S100 fraction, and almost none in the ribosome-high salt wash fraction, indicating
that the protein in active form localizes mainly in the nuclei. Competition experiments with various cap analogues showed
that the G(5′)ppp(5′)N- blocking structure as well as the methyl residue at the N7 position of the blocking guanosine is important
for the binding of this protein, and that the trlmethylguanosine cap structure which exists at the 5′ termini of many snRNAs
is not recognized by this protein. Immunoprecipitation experiments using various antisnRNP antibodies suggested that this
protein is partially associated with U2 snRNP. We purified this protein to near homogeneity from a HeLa nuclear extract by
several chromatographic procedures including capped RNA-Sepharose chromatography. The purified protein shows molecular weight
of 80 kilodaltons, as judged by SDS gel electrophoresis, and binds specifically to the cap structure.
The binding of N-7-substituted cap analogues to eIF-4E from human erythrocytes is described. Data presented here indicate that there is a correlation between the tightness of binding of these cap analogues to eIF-4E and their potency as inhibitors of protein synthesis. This result indicates that the inhibitory activity of the cap analogues is strictly a function of the affinity of the analogue for eIF-4E under equilibrium conditions. The pH dependence of binding of the cap analogues to eIF-4E indicates that the enolate form of the cap is preferred, as originally postulated by Rhoads et al. [(1983) Biochemistry 22, 6084-6088]. Data indicate that there are differences in the mode of binding of alkyl-substituted and aryl-substituted cap analogues to eIF-4E arising from favorable interactions of the phenyl ring with the guanosine moiety. These differences may explain the enhanced recognition of the aryl-substituted cap analogues by eIF-4E.
The gene for the influenza viral PB2 protein, which recognizes and binds the 5'-terminal cap 1 structures (m7GpppNm) on eukaryotic mRNAs, was inserted into a bovine papilloma virus vector under the control of a mouse metallothionein I (MT-I) promoter. After transfection of this vector into mouse NIH 3T3 cells, a cell line, cPB2, was obtained that produces PB2-specific mRNA and authentic PB2 protein. Induction of the MT-I promoter with CdCl2 causes about a 10-fold increase in PB2 mRNA and protein levels. The expressed PB2 protein is functional, as it relieves the block in viral mRNA synthesis exhibited by a temperature-sensitive viral mutant containing a cap-binding defect in the PB2 protein. This demonstrates complementation of a function of a negative-strand RNA virus by a gene product expressed in a cell line from recombinant DNA.
Circular dichroism studies have shown that eukaryotic initiation factor 4E contains low amounts of alpha-helix; the main elements of secondary structure are beta-sheets/turns and aperiodic regions. Interactions with cap analogs are accompanied by small but reproducible changes in overall secondary structure, which may also involve more significant perturbations of localized regions containing certain phenylalanine residues. Dissociation constants for interactions with nucleotides have been established from fluorescence titrations. Results show that the (N-7) methylated guanosine nucleotides bound more strongly than their nonmethylated counterparts. Involvement of a key tryptophan residue in the cap binding site was suggested. Additional studies with two cap binding mutant forms of the protein, designated SK-4 (W----75----L) and SK-6 (W----115----L), confirmed and extended these observations. Fluorescence melting experiments indicated that binding of cap analogs stabilized the protein against thermal perturbation and demonstrated subtle differences in folding between the wild-type and mutant forms of the protein. These subtle differences in folding may account for the observed loss in cap specificity of both mutant forms.
Initiation factor 4E is a 24-kilodalton polypeptide that binds specifically to the 5' cap structure of eukaryotic mRNAs. Sequence analysis of cDNA clones of initiation factor 4E from several species revealed a high tryptophan content (8 residues). Strikingly, all tryptophans are conserved evolutionarily in number and position between yeast and mammals. Here we show, using site-directed mutagenesis, that two of the tryptophans (those referred to as numbers 1 and 8) are absolutely required for the cap binding activity of an Escherichia coli expressed initiation factor 4E.
The 25-kDa mRNA cap-binding protein (CBP) involved in translation was purified by affinity chromatography from human erythrocytes and rabbit reticulocytes. The sequences of eight human and seven rabbit tryptic and V8 proteolytic peptides were determined. Based on the peptide sequence data, oligodeoxynucleotide probes were synthesized and used to screen human fibroblast and lymphocyte lambda cDNA libraries. The DNA sequence obtained from recombinant lambda phage inserts was found to code for all but one peptide. A 23-base oligonucleotide was synthesized based on the DNA sequence and used to prime synthesis of cDNA from human placental mRNA to construct a third library in lambda gt10. Screening with a 22-base oligonucleotide, whose sequence was upstream from the 23-base primer, yielded numerous recombinant phages with approximately equal to 250-base inserts. The 1900-base-pair cDNA sequence compiled from all phage inserts appeared to represent the entire primary sequence of CBP (Mr 25,117). Blot analysis of human placental and HeLa mRNA revealed multiple CBP mRNA species ranging from 1925 to 2250 bases. The amino acid sequence of CBP showed homology to the cap-binding PB2 protein of influenza virus.
We have isolated genomic and cDNA clones encoding protein synthesis initiation factor eIF-4E (mRNA cap-binding protein) of
the yeast Saccharomyces cerevisiae. Their identity was established by expression of a cDNA in Escherichia coli. This cDNA
encodes a protein indistinguishable from purified eIF-4E in terms of molecular weight, binding to and elution from m7GDP-agarose
affinity columns, and proteolytic peptide pattern. The eIF-4E gene was isolated by hybridization of cDNA to clones of a yeast
genomic library. The gene lacks introns, is present in one copy per haploid genome, and encodes a protein of 213 amino acid
residues. Gene disruption experiments showed that the gene is essential for growth.
Three highly purified preparations (preparations I, II-1, and II-2) have been obtained from wheat germ and shown to support in vitro polypeptide synthesis directed by capped or uncapped mRNAs in a eukaryotic initiation factor 4B (eIF-4B)-deficient system. The three preparations differ, however, in polypeptide composition and in the ability to overcome the inhibitory effect of 7-methylguanosine 5'-triphosphate (m7GTP) on in vitro polypeptide synthesis. Preparation I contains two polypeptides (Mr = 80,000 and 28,000), which are present in a 1:1 molar ratio and are associated in a high molecular weight complex. Preparation II-1 contains two major polypeptides (Mr = 220,000 and 26,000) and preparation II-2 also contains two major polypeptides (Mr = 110,000 and 26,000). Preparations II-1 and II-2 are high molecular weight complexes; neither contains detectable amounts of a Mr 80,000 or a Mr 50,000 component. Preparations II-1 and II-2 both overcome m7GTP inhibition, whereas preparation I does not. These findings raise several questions with regard to the identity of eIF-4B and its relationship to cap recognition factors.
The messenger RNA cap-binding protein (CBP) was isolated from human erythrocyte, rabbit erythrocyte, and rabbit reticulocyte lysate by affinity chromatography on 7-methylguanosine 5'-triphosphate-Sepharose. The specific activity of binding to capped oligonucleotides was similar for the human erythrocyte and rabbit reticulocyte CBPs. Isoelectric focusing of human and rabbit preparations revealed that each was composed of up to five species. The pI values of human and rabbit CBPs ranged from 5.7 to 6.5. The predominant form in erythrocytes had a pI of 6.3 while in reticulocytes, two major species, having pI values of 5.9 and 6.3, were present. Labeling of rabbit reticulocytes with [32P]orthophosphate revealed that the pI 5.9 but not the pI 6.3 form contained phosphate. All of the phosphate was found in phosphoserine residues. The amino acid compositions of human erythrocyte and rabbit reticulocyte CBPs were quite similar. Both proteins had 7 tryptophanyl and 6 cysteinyl residues. Labeling with [1-14C]iodoacetic acid under native and denaturing conditions provided evidence that 2 of the cysteinyl residues are present in the reduced form and 4 in disulfide bridges. Species of CBP with faster or slower electrophoretic mobilities could be generated by treatment of the protein either with O2 in the presence of a catalyst or with dithiothreitol. The predominant form of the untreated protein migrated between these two forms.
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.
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 co-migrate 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 QSHADTATKSGSTTKNRF. 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 P, 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.
Eukaryotic translation initiation factor eIF-4E plays a central role in the recognition of the 7-methylguanosine-containing cap structure of mRNA and the formation of initiation complexes during protein synthesis. eIF-4E exists in both phosphorylated and nonphosphorylated forms, and the primary site of phosphorylation has been identified. Previous studies have suggested that eIF-4E phosphorylation facilitates its participation in protein synthesis. However, the biochemical basis for the functional difference between the two forms of eIF-4E is unknown. To address this directly, we have developed a method for the separation of phosphorylated and nonphosphorylated eIF-4E from rabbit reticulocytes by chromatography on rRNA-Sepharose. Using the resultant purified forms, we have studied the protein's interaction with the cap analogs m7GTP and m7GpppG and with the cap of globin mRNA by fluorescence quenching of tryptophan residues. It was found that phosphorylated eIF-4E had 3- to 4-fold greater affinity for cap analogs and mRNA than nonphosphorylated eIF-4E. The equilibrium binding constants (x 10(5), expressed as M-1) for the interaction of phosphorylated eIF-4E with m7GTP, m7GpppG, and globin mRNA were 20.0 +/- 0.1, 16.4 +/- 0.1, and 31.0 +/- 0.1, respectively, whereas those for the nonphosphorylated form were 5.5 +/- 0.4, 4.3 +/- 0.4, and 10.0 +/- 0.1, respectively. Treatment with potato acid phosphatase converted the phosphorylated form to the nonphosphorylated form and decreased the binding constant for m7GTP by a factor of 3. The increased affinity for mRNA caps may account for the in vivo and in vitro correlations between eIF-4E phosphorylation and accelerated protein synthesis and cell growth.
This article is a review of what is known about the 7-methylguanosine-containing “cap” structure of eukaryotic messenger RNA and its participation in the initiation of protein synthesis. Particular attention will be paid to the protein which is thought to mediate the entry of mRNA into the cycle of protein synthesis by recognizing the cap structure, termed cap-binding protein (CBP). Previous review articles dealing with this or related topics include those of Shatkin (1976), Banerjee (1980), Ehrenfeld (1982), Penman (1982) and Nielsen et al. (1983). The primary focus of this review will be translational events. Topics which will not be treated include biosynthesis of caps (Rottman 1978; Banerjee 1980), the role of the cap structure in stabilization of mRNA against degradation (Shimotohno et al. 1977; Furuichi et al. 1977), and the CBP which is involved in initiation of transcription of influenza virus (Blaas et al. 1982).
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
As a model study to investigate the sequence dependence of a peptide for the interaction with nucleic acid base, four kinds of tryptophan-containing dipeptides [Trp-Glu, Glu-Trp, Trp-Gly and Gly-Trp] were synthesized, and their abilities in forming the complexes with guanine base were examined by fluorescence and proton nuclear magnetic resonance (1H-NMR) methods. The fluorescence titration of each dipeptide with 7-methylguanosine-5'-phosphate (m7GMP) indicated that although the stacking interaction dominates the binding in each peptide, the carboxyl side chain of Glu plays an additional role in the binding with the base. The effect of Glu residue and its sequence dependence for the interaction was more clearly demonstrated by the 1H-NMR titration. Since the association constants determined from the downfield shift of the guanine NH2 resonance exhibited the same tendency as those from the upfield shift of the guanine N7-methyl protons [Trp-Glu > Trp-Gly >Glu-Trp > Gly-Trp], the close cooperation between the hydrogen bond and stacking interactions was suggested to be important for the tight binding of the peptides to the guanine base. Further, it was suggested that the peptide which contains an aromatic amino acid at the N-terminal side and an acidic one at the C-terminal side has an advantage in forming such a complex.
As a model to investigate the mode of recognition of the base guanine by peptides and proteins, the crystal structure of the 7-methylguanosine-5′-phosphate–tryptophanylglutamic acid complex was analysed by X-ray diffraction; this is the first crystal structure determination of a peptide–nucleotide complex. The complex crystals are stabilized by extensive hydrogen-bond formation in which three independent water molecules per complex pair participate. Both molecules are joined by the coupled contributions of the triple hydrogen bonds between the guanine base and the peptide backbone chain and of the prominent stacking interactions between the guanine base and the tryptophan indole side-chain, suggesting the importance of the coupling of hydrogen bonding and stacking interactions for recognition of the base to occur.
Four mutants of the human cap binding protein (hCBP), in which Trp-102, Glu-103, Asp-104 or Glu-105 was changed to the aliphatic Leu or Ala, were prepared, and their cap binding abilities were examined. Cap binding abilities of two mutants. W102L (Trp-102→Leu) and E105A (Glu-105→Ala), were significantly decreased in comparison with the wild-type hCBP. This result suggest that Trp-102 and Glu-105 are both necessary for the cap binding, and the most probable binding mode with the m7G of cap structure is the combination of the stacking by Trp-102 and the hydrogen-bond pairing by Glu-105, as was already proposed from the model studies.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
The stacking and hydrogen bonding abilities of Trp-(Gly)n-Glu (n = 0 approximately 3) for the interaction with 7-methylguanine (m7G) base were examined by fluorescence and 1H-NMR methods, and it was shown that they correlate with the distance between the Trp and Glu residues, and become most significant when both residues are separated from each other by two Gly residues (n = 2). Based on this insight, the sequence conserved between the human and yeast cap binding proteins (CBPs) was surveyed, and the sequence of Trp-Glu-Asp-Glu (No. 102-105 in human CBP) was selected as a probable site for the binding with mRNA cap structure. Thus, the stacking and hydrogen bonding abilities of Trp-Glu-Asp-Glu with m7G cap structure were examined by comparative experiments using its analogous peptides. The results showed that the fourth Glu residue is important not only for the construction of hydrogen bond pairing with m7G base but also for strengthening the stacking interaction between the Trp indole ring and m7G base. Taking account of the recognition analysis using the mutant CBP proteins by site-directed mutagenesis (Ueda, H., Iyo, H., Doi, M., Inoue, M., Ishida, T., Morioka, H., Tanaka, T., Nishikawa, S. and Uesugi, S. (1991) FEBS Lett. 280, 207-210), this cooperative interaction could be important for the recognition of mRNA cap structure.
An artificial gene coding for the human cap binding protein (hCBP: human IF-4E) was chemically synthesized and expressed in Escherichia coli under the control of a trp promoter. The DNA duplex of 662 bp was designed and constructed from 44 oligodeoxynucleotide fragments of typically 30 nucleotides in length. Although the hCBP gene was not directly expressed in E. coli HB101, we succeeded in its high-level expression as a fusion protein connected with a portion of human growth hormone through a tetradecapeptide (Asp-Asp-Pro-Pro-Thr-Val-Glu-Leu-Gln-Gly-Leu-Val-Pro-Arg) that contains the recognition sequence for a site-specific protease alpha-thrombin. Upon induction with 3-indoleacrylic acid, the fusion protein accumulated with a yield of about 20% of the total proteins of the host cell. Upon the treatment of the fusion protein with alpha-thrombin, which recognizes the sequence "Val-Pro-Arg," specific proteolysis at the fused junction occurred efficiently. In this system, nonspecific digestion by alpha-thrombin was not marked. About 15 mg of recombinant hCBP was obtained from a 1-liter culture. Association constants between the recombinant hCBP and mRNA cap structure analogues were determined by fluorescence spectroscopy. The values obtained for the m7GpppA, m7GTP, and m7GMP were almost the same as those reported for the IF-4E isolated from human erythrocyte cells.(ABSTRACT TRUNCATED AT 250 WORDS)
This chapter discusses the basic technique for synthesizing long, capped transcripts in vitro by SP6 and T7 RNA polymerases. The sequence to be transcribed is cloned into the appropriate vector. The transcription reaction consists of a linear DNA template, ribonucleotide triphosphates, RNA polymerase, and, generally, an RNase inhibitor in a relatively simple buffer. An increasing number of plasmid vectors have been constructed to facilitate the cloning of sequences to be transcribed in vitro. Plasmids containing the cloned DNA sequence can be grown and purified using many techniques. Because supercoiled plasraids containing a bacteriophage promoter are very efficiently transcribed, yielding large, often multimeric transcripts, it is very important to cleave the entire template to completion. RNA transcripts containing modified nucleotides have been used in a variety of ways. RNAs containing biotinylated nucleotides can be used as nonradioactive probes in any sort of hybridization assay in place of DNA probes.
The binding of analogues of the 7-methylguanosine-containing cap, m7GTP and m7GpppG, to eIF-4E from human erythrocytes as a function of pH, temperature, and ionic strength is described. From the pH-dependent binding of m7GTP and m7GpppG to eIF-4E, a new model describing the nature of the cap.eIF-4E interaction is proposed. The thermodynamic values and ionic strength dependence of binding are consistent with a binding site which is primarily hydrophobic. Fluorescence and circular dichroism data indicate that tryptophan residues may be involved in base-stacking interactions with the cap in a somewhat buried environment. The model presented here confirms the earlier proposal [Rhoads et al. (1983) Biochemistry 22, 6084-6088] that the enolate tautomer of the cap is preferred for interaction and further proposes that the interaction is with a protonated amino acid residue, such as histidine, while stacking with an aromatic amino acid, such as tryptophan.
Several features of the 5´ noncoding region of eukaryotic mRNAs are of importance for translational control at the initiation level; these include the 5´ cap structure and the 5´ noncoding region (comprising primary and secondary structure determinants). Various studies suggest that the cap structure mediates the ATP dependent melting of the 5´ secondary structure of mRNA through the activity of a cap-binding-protein complex, in conjunction with other initiation factors required for mRNA binding. A model explaining these activities is presented in this chapter. It also discusses the possibility of internal binding of ribosomes to eukaryotic mRNAs in the light of recent evidence. Recent in vitro studies demonstrate that the cap structure may also function in nuclear processes, including pre-mRNA splicing, and processing of the 3´ end. In addition, the cap structure serves to stabilize mRNAs in the cytoplasm and pre-mRNAs in the nucleus against 5´ exonucleolytic degradation. It has been concluded that despite the advances in understanding the mechanism of function of the initiation factors involved in mRNA binding to ribosomes, it is unclear how and what the ribosome recognizes on the mRNA for its initial binding.
The conformation of 7-methylguanosine 5′-monophosphate (m7GMP) and its interaction with L-phenylalanine (Phe) have been investigated
by X-ray crystallographic, H-nuclear magnetic resonance, and energy calculation methods. The N(7) methylation of the guanine
base shifts m7GMP toward an anti—gauche, gauche conformation about the glycosyl and exocyclic C(4′)–C(5′) bonds, respectively. The prominent stacking observed between the
benzene ring of Phe and guanine base of m7GMP is primarily due to the N(7) quarternization of the guanine base. The formation
of a hydrogen bonding pair between the anionic carboxyl group and the guanine base further stabilizes this stacking interaction.
The present results imply the importance of aromatic amino acids as a hallmark for the selective recognition of a nucleic
acid base.
Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung+) Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.
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Stacking interactions were shown by spectroscopic and X-ray crystallographic studies to be formed between the tryptophan and the protonated 7-methylguanine derivatives. These interactions would be in part responsible for the specific interaction between the 5'-terminal capped structure of mRNA and its binding protein.
A Drosophila melanogaster cDNA clone encoding the translation initiation factor eIF-4E was isolated and sequenced. The deduced polypeptide consists of 259 amino acids with a predicted molecular weight of 29,223. It shares 48%, 37% and 35% identity to its mammalian, yeast and wheat counterparts, respectively. Several residues (including eight tryptophans), which were shown to be critical for the function of mammalian and yeast eIF-4Es, are conserved in the Drosophila protein. Three transcripts of the eIF-4E gene were detected throughout Drosophila development.
In order to obtain the active form of recombinant human initiation factor (eIF) 4E effectively, an artificial synthetic gene was cloned into an expression vector (pMAL-p2) and the soluble expression was attempted in Escherichia coli under the control of a tac promoter. Two expression systems were finally constructed as a fusion protein with maltose-binding protein, which contain a recognition sequence for the site specific protease alpha-thrombin and factor Xa, respectively. Most of the fusion protein was induced as a soluble form. The soluble human eIF-4E digested from the fusion protein showed binding specificity for the m7GTP affinity column.
An artificial synthetic gene coding for human eIF-4E was cloned into an expression vector and direct expression was attempted in Escherichia coli [BL21(DE3) strain] under the control of T7 promoter. The active gene product which was induced in high yield (ca. 4 mg/100 ml) by isopropyl-beta-D-thiogalactopyranoside was purified to homogeneity by a two-step chromatographic procedure with a good yield (ca. 74%), and was confirmed to be recombinant human eIF-4E by amino acid composition and sequence analyses, isoelectric focusing, and absorption spectral measurements. The identity of three-dimensional structures between the recombinant and native human eIF-4Es was confirmed by CD and fluorescence measurements.
The cloning is described of two related human complementary DNAs encoding polypeptides that interact specifically with the translation initiation factor eIF-4E, which binds to the messenger RNA 5'-cap structure. Interaction of these proteins with eIF-4E inhibits translation but treatment of cells with insulin causes one of them to become hyperphosphorylated and dissociate from eIF-4E, thereby relieving the translational inhibition. The action of this new regulator of protein synthesis is therefore modulated by insulin, which acts to stimulate the overall rate of translation and promote cell growth.
Recombinant human eukaryotic initiation factor-4E (eIF-4E), purified by m7GTP-Sepharose 4B affinity chromatography, was used for crystallization. After concentration of the eIF-4E protein (7 mg/ml), the solution was subjected to crystallization by the hanging-drop method. Transparent needle crystals complexed with m7GTP were obtained from 50 mM 2-(N-morpholino)ethanesulfonic acid-KOH buffer (pH 6.5) containing 25% (w/v) polyethylene glycol 6000 and 0.2 M (NH4)2SO4. The crystals belong to tetragonal space group P4(1) or P4(3), of Z = 4, with unit-cell dimensions of a = 89.26, b = 89.26, and c = 38.51 angstrum, and diffract beyond 2.1 angstrum resolution. The Vm value was calculated to be 3.07 angstrum 3/Da, which indicates a solvent content of 59.9%.
- N Sonenberg
- R E Rhoads
Sonenberg, N. (1988) Prog. Nucleic Acid Res. Mol. Biol. 35, 173-Rhoads, R. E. (1993) J. Bid. Chem. 268, 3017-3020.
- T Ishida
- H Iyo
- H Ueda
- M Doi
- M Inoue
- S Nishimura
- K Kitamura
Ishida, T., Iyo, H., Ueda, H., Doi, M., Inoue, M., Nishimura, S. &
Kitamura, K. (1991) J. Chem. SOC. Perkin Trans. I, 1847-1853.
- R E Rhoads
Rhoads, R. E. (1985) Prog. Mol. Subcell. Bid. 9, 104-155.
- H Iyo
- H Ueda
- Y Usami
- Y Kafuku
- M Doi
- M Inoue
- T Ishida
Iyo, H., Ueda, H., Usami, Y., Kafuku, Y., Doi, M., Inoue, M. &
Ishida, T. (1991) Chem. Pharm. Bull. 39, 2483-2486.
- G Hernandez
- J M Sierra
- T Ishida
- M Katsuta
- M Inoue
- Y Yamagata
- K Tomita
- S E Carberry
- R E Rhoads
- D J Goss
Hernandez, G. & Sierra, J. M. (1995) Biochim. Biophys. Actu 1261,
Ishida, T., Katsuta, M., Inoue, M., Yamagata, Y. & Tomita, K. (1983)
Carberry, S. E., Rhoads, R. E. & Goss, D. J. (1989) Biochemistv
28, 8078-8083.
- M Ohno
- N Kataoka
- Y Shimura
Ohno, M., Kataoka, N. & Shimura, Y. (1990) Niicleic Acids Res.
(1994) J. Biochem. (Tokyo) 116, 687-693.
- B Joshi
- A Cai
- B D Keiper
- W B Minich
- R Mendoz
- C M Beach
- J Stepinski
- R Stolarski
- E Darzynkiewicz
- R E Rhoads
Joshi, B., Cai, A,, Keiper, B. D., Minich, W. B., Mendoz, R., Beach,
C. M., Stepinski, J., Stolarski, R., Darzynkiewicz, E. & Rhoads,
R. E. (1995) J. Bid. Chem. 270, 14597-14603.
- M Altmann
- I Edery
- H Trachsel
- N Sonenberg
Altmann, M., Edery, I., Trachsel, H. & Sonenberg, N. (1988) J. Biol.
- N Sonenberg
Sonenberg, N. (1988) Prog. Nucleic Acid Res. Mol. Biol. 35, 173Rhoads, R. E. (1993) J. Bid. Chem. 268, 3017-3020.
- M Altmann
- C Handschin
- H Trachsel
Altmann, M., Handschin, C. & Trachsel, H. (1987) Mol. Cell. B i d.
- H Ueda
- H Iyo
- M Doi
- M Inoue
- T Ishida
- H Moriokd
- T Tanaka
- S Nishikawa
- S Uesugi
Ueda, H., Iyo, H., Doi, M., Inoue, M., Ishida, T., Moriokd, H., Tanaka, T., Nishikawa, S. & Uesugi, S. (1991) FEBS Lett. 280,
- W D Mccubbin
- I Edery
- M Altmann
- N Sonenberg
- C M Kay
McCubbin, W. D., Edery, I., Altmann, M., Sonenberg, N. & Kay,
C. M. (1988) J. B i d. Chern. 263, 17663-17671.
- J K Yisraeli
- D A Melton
Yisraeli, J. K. & Melton, D. A. (1989) Methods Enzymol. 180, 42-