Low Affinity and Slow Na+ Binding Precedes High Affinity Aspartate Binding in the Secondary-active Transporter GltPh

Glt_Ph from Pyrococcus horikoshii is a homotrimeric Na⁺-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters that take up the neurotransmitter glutamate. Each protomer in Glt_Ph consists of a trimerization domain involved in subunit interactions and a transport domain containing the substrate binding site. Here, we have studied the dynamics of Na⁺ and aspartate binding to Glt_Ph. Tryptophan fluorescence measurements on the fully active single tryptophan mutant F273W revealed that Na⁺ binds with low affinity to the apoprotein (K_d 120 mm), with a particularly low k_on value (5.1 m⁻¹s⁻¹). At least two sodium ions bind before aspartate. The binding of Na⁺ requires a very high activation energy (E_a 106.8 kJ mol⁻¹) and consequently has a large Q₁₀ value of 4.5, indicative of substantial conformational changes before or after the initial binding event. The apparent affinity for aspartate binding depended on the Na⁺ concentration present. Binding of aspartate was not observed in the absence of Na⁺, whereas in the presence of high Na⁺ concentrations (above the K_d for Na⁺) the dissociation constants for aspartate were in the nanomolar range, and the aspartate binding was fast (k_on of 1.4 × 10⁵ m⁻¹s⁻¹), with low E_a and Q₁₀ values (42.6 kJ mol⁻¹ and 1.8, respectively). We conclude that Na⁺ binding is most likely the rate-limiting step for substrate binding.     

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.     

Evidence for Variation in the Optimal Translation Initiation Complex: Plant eIF4B,eIF4F,and eIF(iso)4F Differentially Promote Translation of mRNAs

Expansins are cell wall proteins associated with the process of plant growth. However, investigations in which expansin gene expression has been manipulated throughout the plant have often led to inconclusive results. In this article, we report on a series of experiments in which overexpression of expansin was targeted to specific phases of leaf growth using an inducible promoter system. The data indicate that there is a restricted window of sensitivity when increased expansin gene expression leads to increased endogenous expansin activity and an increase in leaf growth. This phase of maximum expansin efficacy corresponds to the mid phase of leaf growth. We propose that the effectiveness of expansin action depends on the presence of other modulating factors in the leaf and we suggest that it is the control of expression of these factors (in conjunction with expansin gene expression) that defines the extent of leaf growth. These data help to explain some of the previously observed variation in growth response following manipulation of expansin gene expression and highlight a potential linkage of the expression of modifiers of expansin activity with the process of exit from cell division.Expansins were initially identified as cell wall proteins that had the ability to promote the extension of plant tissue in vitro (McQueen-Mason et al., 1992). Further work on these proteins and the genes encoding them has revealed a picture in which, although a general correlation with growth has often been substantiated, it is clear that control of growth is a much more complex process than the control of expression of a single protein type (for review, see Cosgrove, 2000; Lee et al., 2001; Li et al., 2003). In addition, although it is clear that expansins play a role in many growth processes, there are a number of open questions about exactly how expansins contribute to these processes. First, we still have a very limited understanding of the molecular mechanism of expansin action. Efforts to identify classical enzymatic activities associated with expansins have proven fruitless (McQueen-Mason and Cosgrove, 1995; Li and Cosgrove, 2001) and the remaining, somewhat speculative, interpretation is that expansins intercalate within carbohydrate matrices in the cell wall, leading to transient loosening of noncovalent interactions and, thus, the ability of these matrices to move relative to each other (McQueen-Mason and Cosgrove., 1994). In addition, by unlocking aspects of the molecular architecture of the cell wall, expansins may allow access of other cell wall proteins/enzymes to particular substrates. Depending on the nature of these other proteins/enzymes, expansin activity could thus be associated not only with growth processes, but also with cell wall modifications linked with differentiation. Such a mechanism would help to explain observations (described below) that the effectiveness of expansin action appears to be context dependent and is not only associated with changes in plant growth but also with differentiation.Various analyses have revealed that expansins are present in a wide range of plants, including bryophytes, ferns, angiosperms, and conifers (Hutchison et al., 1999; Kim et al., 2000; Schipper et al., 2002). Moreover, they are generally encoded by relatively large gene families whose members often show distinct patterns of gene expression (Kende et al., 2004). Some of these expression patterns correlate with growth processes, such as root growth (Wu et al., 1996), internode growth (Cho and Kende, 1997), leaf growth (Muller et al., 2007), and cotton (Gossypium hirsutum) fiber growth (Ruan et al., 2001), whereas others correlate with events of differentiation, such as fruit ripening (Rose et al., 1997; Brummell et al., 1999b), grass tiller formation (Reidy et al., 2001), and endosperm breakdown (Chen and Bradford, 2000). In addition, some novel nonplant expansin activities have been identified that suggest that pathogens may induce altered cell wall structure via an expansin-mediated mechanism (Qin et al., 2004). Since in vitro assays have suggested that the activities of expansins extracted from different sources tend to be similar (Cosgrove, 2000), it has been proposed that this tissue, organ, and environmental specificity of expression pattern reflects a specialized role for expansins in specific contexts rather than any major difference in activity of the protein. As stated above, this specific function may depend on the presence (or absence) of tissue-specific cofactors, the nature of which is as yet unclear.In addition to biochemical approaches to understanding expansin function, numerous groups have undertaken transgenic experiments to alter expansin gene expression in plants to observe the outcome on plant phenotype. Although some successes with antisense strategies have been reported (Brummell et al., 1999a; Cho and Cosgrove, 2000), the encoding of expansin by large gene families means that genetic redundancy poses a significant problem for such approaches (e.g. Schipper et al., 2002). Simple overexpression strategies to alter expansin activity may also be difficult to interpret. For example, when expansins were constitutively overexpressed throughout Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), and rice (Oryza sativa) plants, the outcomes tended to be pleiotropic, including a decrease in overall plant growth (Cho and Cosgrove, 2000; Rochange et al., 2001; Choi et al., 2003). However, when altered expansin expression was targeted more specifically to a particular tissue or organ, then more easily interpretable results were obtained. For example, when altered expansin expression was directed to the developing leaf petiole in Arabidopsis, altered leaf growth was observed (Cho and Cosgrove, 2000), consistent with the idea that expansins promote growth, and when inducible expression of expansin was targeted throughout rice plants, quantitative changes in growth were observed (Choi et al., 2003). The results of these experiments indicate that expansin gene expression can be used as a tool to modulate growth, but that the timing and spatial extent of expression can have a significant influence on the phenotype observed. Again, these data support the hypothesis that the effectiveness of expansin in promoting specific growth or differentiation events is dependent on the presence of particular tissue- or developmental-specific cofactors. So far, little progress has been made on the identification and characterization of these cofactors.In previous work, we reported on the characterization of transgenic lines of tobacco (Nicotiana tabacum) in which a cucumber (Cucumis sativus) expansin (CsEXP1) could be induced by application of a chemical inducer (anhydrotetracycline [Ahtet]). In these experiments, we targeted expansin overexpression to localized regions of either the shoot apical meristem or very young leaf primordia, which led to localized promotion of growth (Pien et al., 2001), consistent with the idea that expansins play a role in the endogenous mechanism of leaf initiation (Reinhardt et al., 1998). However, when inductions were performed throughout the plant the resulting phenotypes were variable and difficult to interpret (S. Pien and A. Fleming, unpublished data), in line with other reports (Rochange et al., 2001). To investigate the possibility that this variable response reflected a differential sensitivity to expansin in different tissues at different stages of development, we performed a series of experiments (reported here) in which overexpression of expansin was targeted to specific stages of leaf growth. Our data indicate that the efficacy of expansin action depends on the presence of other factors that are present in a developmentally controlled fashion, so that increased expansin gene expression is only effective in promoting leaf growth during a specific developmental period of leaf growth. This period corresponds to the inflection point of relative growth rate (RGR) and, thus, to the phase of maximum leaf growth rate. An intriguing article by Cookson et al. (2005) reported on potential correlations between various parameters of leaf growth and final leaf size. They found that the best predictor of final leaf size was the maximum value of absolute leaf growth rate. Thus, the experiments reported here identify a novel, developmental control of expansin efficacy in the regulation of leaf growth, investigate the reported correlation between maximal leaf expansion rate and leaf size, and provide an insight into potential means of controlling leaf growth.     

Requirement of RNA Binding of Mammalian Eukaryotic Translation Initiation Factor 4GI (eIF4GI) for Efficient Interaction of eIF4E with the mRNA Cap

Eukaryotic mRNAs possess a 5′-terminal cap structure (cap), m⁷GpppN, which facilitates ribosome binding. The cap is bound by eukaryotic translation initiation factor 4F (eIF4F), which is composed of eIF4E, eIF4G, and eIF4A. eIF4E is the cap-binding subunit, eIF4A is an RNA helicase, and eIF4G is a scaffolding protein that bridges between the mRNA and ribosome. eIF4G contains an RNA-binding domain, which was suggested to stimulate eIF4E interaction with the cap in mammals. In Saccharomyces cerevisiae, however, such an effect was not observed. Here, we used recombinant proteins to reconstitute the cap binding of the mammalian eIF4E-eIF4GI complex to investigate the importance of the RNA-binding region of eIF4GI for cap interaction with eIF4E. We demonstrate that chemical cross-linking of eIF4E to the cap structure is dramatically enhanced by eIF4GI fragments possessing RNA-binding activity. Furthermore, the fusion of RNA recognition motif 1 (RRM1) of the La autoantigen to the N terminus of eIF4GI confers enhanced association between the cap structure and eIF4E. These results demonstrate that eIF4GI serves to anchor eIF4E to the mRNA and enhance its interaction with the cap structure.The cap structure, m⁷GpppN, is present at the 5′ terminus of all nuclear transcribed eukaryotic mRNAs. Cap-dependent binding of the ribosome to mRNA is mediated by the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E), which forms a complex termed eIF4F together with eIF4G and eIF4A. Mammalian eIF4G, which has two isoforms, eIF4GI and eIF4GII, is a modular, multifunctional protein that binds to poly(A)-binding protein (PABP) (14) and eIF4E (18, 20) via the N-terminal third region. Mammalian eIF4G binds to eIF4A and eIF3 (15) via the middle third region and to eIF4A and Mnk protein kinase at the C-terminal region. eIF4GI also possesses an RNA-binding sequence (2, 9, 33) in the middle region. There are two RNA-binding sites on eIF4GI; one is located amino terminal to the first HEAT domain, and the other is located within the first HEAT domain (23). Mammalian and Saccharomyces cerevisiae eIF4E are similar in size (24 kDa), but mammalian eIF4GI (220 kDa) is larger than its yeast counterpart (150 kDa), as the latter lacks a C-terminal domain corresponding to mammalian eIF4GI (38).The affinity of eIF4E for the cap structure has been a matter of dispute for some time. The earlier works of Carberry et al. (4) and Ueda et al. (39) estimated the equilibrium dissociation constant (K_d) of the eIF4E-cap complex by fluorescence titration to be 2 × 10⁻⁶ to 5 × 10⁻⁶ M depending on the nature of the cap analog. Later on, development of a new methodology for the fluorescence titration experiments yielded K_d values of 10⁻⁷ to 10⁻⁸ (29, 41). The source of the difference with the previous reports was thoroughly analyzed (29, 30). The interaction between the cap structure and eIF4E is dramatically enhanced by eIF4GI. This was first reported by showing that cross-linking of mammalian eIF4E to the cap structure is more efficient when it is a subunit of the eIF4F complex (19) or when it is complexed to eIF4GI (11). A similar enhancement of the binding of eIF4E to the cap structure was observed in yeast (40). However, two very different mechanisms were proposed to explain these observations. For the mammalian system, it was postulated that the middle segment of eIF4GI, which binds RNA, stabilizes the eIF4E interaction with the cap structure (11). This model was based primarily on the finding that in poliovirus-infected cells, eIF4GI is cleaved between its N-terminal third and the middle third, and consequently, eIF4E remains attached to the N-terminal eIF4GI fragment lacking the RNA-binding region. Under these conditions, cross-linking of eIF4E to the cap structure was poor (19, 31). In contrast, in yeast, a strong interaction between the cap structure and eIF4E was achieved using an eIF4G fragment containing the eIF4E-binding site that lacks the RNA-binding region (34, 40). Also, the yeast eIF4G fragment from amino acids 393 to 490 (fragment 393-490), which does not contain the RNA-binding site, forms a right-handed helical ring that wraps around the N terminus of eIF4E. This conformational change was suggested in turn to engender an allosteric enhancement of the association of eIF4E with the cap structure (10). Such an interaction between mammalian eIF4GI and eIF4E has not been reported.To understand the mechanism by which eIF4GI stimulates the interaction of eIF4E with the cap structure in mammals, we reconstituted the eIF4E-cap recognition activity in vitro with purified eIF4E and eIF4GI recombinant proteins. Using a chemical cross-linking assay, we demonstrate that only mammalian eIF4GI fragments possessing RNA-binding activity enhance the cross-linking of eIF4E to the cap structure. Our data provide new insight into the mechanism of cap recognition by the eIF4E-eIF4GI complex.     

Sortase D Forms the Covalent Bond That Links BcpB to the Tip of Bacillus cereus Pili

Bacillus cereus and other Gram-positive bacteria elaborate pili via a sortase D-catalyzed transpeptidation mechanism from major and minor pilin precursor substrates. After cleavage of the LPXTG sorting signal of the major pilin, BcpA, sortase D forms an amide bond between the C-terminal threonine and the amino group of lysine within the YPKN motif of another BcpA subunit. Pilus assembly terminates upon sortase A cleavage of the BcpA sorting signal, resulting in a covalent bond between BcpA and the cell wall cross-bridge. Here, we show that the IPNTG sorting signal of BcpB, the minor pilin, is cleaved by sortase D but not by sortase A. The C-terminal threonine of BcpB is amide-linked to the YPKN motif of BcpA, thereby positioning BcpB at the tip of pili. Thus, unique attributes of the sorting signals of minor pilins provide Gram-positive bacteria with a universal mechanism ordering assembly of pili.Sortases catalyze transpeptidation reactions to assemble proteins in the envelope of Gram-positive bacteria (1). Secreted proteins require a C-terminal sorting signal for sortase recognition such that sortase cleaves the substrate at a short peptide motif and forms a thioester-linked intermediate to its active site cysteine (2–4). Nucleophilic attack by an amino group within the bacterial envelope resolves the thioester intermediate, generating an amide bond tethering surface proteins at their C terminus onto Gram-positive bacteria (5). Four classes of sortases can be distinguished on the basis of sequence homology and substrate recognition (6, 7). Sortase A cleaves secreted protein at LPXTG sorting signals and recognizes the amino group of lipid II peptidoglycan precursors as a nucleophile (8, 9). Sortase B cleaves protein substrates at NPQTN sorting signals (10). This enzyme immobilizes proteins within fully assembled cell walls, utilizing the cell wall cross-bridge as a nucleophile (11). Sortase C cuts LPNTA sorting signals and anchors proteins to the peptidoglycan cross-bridges in sporulating bacteria (12, 13). Finally, sortase D catalyzes transpeptidation reactions in the assembly of pili (14, 15). Sortase D recognizes the amino group of lysine residues within the YPKN motif of pilin subunits as nucleophiles (16). The resultant sortase D-catalyzed amide bond links adjacent pilin subunits to grow the pilus fiber (16, 17).Pili of Gram-positive bacteria comprised either two or three different pilin subunits synthesized as cytoplasmic precursors with N-terminal signal peptides and C-terminal sorting signals (P1 precursors) (14, 18). After translocation across the plasma membrane, P2 precursor species arise from removal of the signal peptide from P1 precursors by a signal peptidase (16). Bacillus cereus pili are composed of two subunits; that is, the major pilin, BcpA, and the minor pilin, BcpB (15). In contrast to BcpA, which is deposited throughout the pilus, BcpB is found at fiber tip (15). Sortase D cleaves the BcpA LPXTG motif sorting signal between the threonine and glycine residues to form an amide bond to the ε-amino group of the lysine within the YPKN motif of adjacent BcpA subunits (16). However, sortase A also cleaves BcpA precursors, which are subsequently linked to the side chain amino group of meso-diaminopimelic acid within lipid II (19). The latter reaction serves to terminate fiber elongation, immobilizing BcpA pili in the cell wall envelope (19).The conservation of sortase D, the YPKN motif, and C-terminal sorting signal in major pilin subunits suggest a universal pilus assembly mechanism among Gram-positive bacteria (14, 20). However, the molecular mechanism whereby bacilli deposit BcpB, the minor pilin, at the tip of BcpA pili is not known. Although the BcpB precursor harbors an N-terminal signal peptide and a C-terminal IPNTG sorting signal, it lacks the YPKN pilin motif of the major subunit (15). Furthermore, the substrate properties of the BcpB IPNTG sorting signal for the four classes of sortases expressed by bacilli has yet to be established.     

NADP+ Binding to the Regulatory Subunit of Methionine Adenosyltransferase II Increases Intersubunit Binding Affinity in the Hetero-Trimer

Mammalian methionine adenosyltransferase II (MAT II) is the only hetero-oligomer in this family of enzymes that synthesize S-adenosylmethionine using methionine and ATP as substrates. Binding of regulatory β subunits and catalytic α2 dimers is known to increase the affinity for methionine, although scarce additional information about this interaction is available. This work reports the use of recombinant α2 and β subunits to produce oligomers showing kinetic parameters comparable to MAT II purified from several tissues. According to isothermal titration calorimetry data and densitometric scanning of the stained hetero-oligomer bands on denatured gels, the composition of these oligomers is that of a hetero-trimer with α2 dimers associated to single β subunits. Additionally, the regulatory subunit is able to bind NADP⁺ with a 1∶1 stoichiometry, the cofactor enhancing β to α2-dimer binding affinity. Mutants lacking residues involved in NADP⁺ binding and N-terminal truncations of the β subunit were able to oligomerize with α2-dimers, although the kinetic properties appeared altered. These data together suggest a role for both parts of the sequence in the regulatory role exerted by the β subunit on catalysis. Moreover, preparation of a structural model for the hetero-oligomer, using the available crystal data, allowed prediction of the regions involved in β to α2-dimer interaction. Finally, the implications that the presence of different N-terminals in the β subunit could have on MAT II behavior are discussed in light of the recent identification of several splicing forms of this subunit in hepatoma cells.     

Selective modification of eukaryotic initiation factor 4F (eIF4F) at the onset of cell differentiation: recruitment of eIF4GII and long-lasting phosphorylation of eIF4E

mRNA translation is mainly regulated at the level of initiation, a process that involves the synergistic action of the 5' cap structure and the 3' poly(A) tail at the ends of eukaryotic mRNA. The eukaryote initiation factor 4G(eIF4G) is a pivotal scaffold protein that forms a critical link between mRNA cap structure, poly(A) tail, and the small ribosomal subunit. There are two functional homologs of eIF4G in mammals, the original eIF4G, renamed eIF4GI, and eIF4GII that functionally complements eIF4GI. To date, biochemical and functional analysis have not identified differential activities for eIF4GI and eIF4GII. In this report, we demonstrate that eIF4GII, but not eIF4GI, is selectively recruited to capped mRNA at the onset of cell differentiation. This recruitment is coincident with a strong and long-lasting phosphorylation of eIF4E and the release of 4E-BP1, a suppressor of eIF4E function, from the cap structure, without a concomitant change in 4E-BP1's phosphorylation. Our data further indicate that cytokines such as thrombopoietin can differentially regulate eIF4GI/II activities. These results provide the first evidence that eIF4GI/II does fulfill selective roles in mammalian cells.     

Inhibition of mTORC1 Enhances the Translation of Chikungunya Proteins via the Activation of the MnK/eIF4E Pathway

Chikungunya virus (CHIKV), the causative agent of a major epidemic spanning five continents, is a positive stranded mRNA virus that replicates using the cell’s cap-dependent translation machinery. Despite viral infection inhibiting mTOR, a metabolic sensor controls cap-dependent translation, viral proteins are efficiently translated. Rapalog treatment, silencing of mtor or raptor genes, but not rictor, further enhanced CHIKV infection in culture cells. Using biochemical assays and real time imaging, we demonstrate that this effect is independent of autophagy or type I interferon production. Providing in vivo evidence for the relevance of our findings, mice treated with mTORC1 inhibitors exhibited increased lethality and showed a higher sensitivity to CHIKV. A systematic evaluation of the viral life cycle indicated that inhibition of mTORC1 has a specific positive effect on viral proteins, enhancing viral replication by increasing the translation of both structural and nonstructural proteins. Molecular analysis defined a role for phosphatidylinositol-3 kinase (PI3K) and MAP kinase-activated protein kinase (MnKs) activation, leading to the hyper-phosphorylation of eIF4E. Finally, we demonstrated that in the context of CHIKV inhibition of mTORC1, viral replication is prioritized over host translation via a similar mechanism. Our study reveals an unexpected bypass pathway by which CHIKV protein translation overcomes viral induced mTORC1 inhibition.     

Translation Directed by Hepatitis A Virus IRES in the Absence of Active eIF4F Complex and eIF2

Translation directed by several picornavirus IRES elements can usually take place after cleavage of eIF4G by picornavirus proteases 2A^pro or L^pro. The hepatitis A virus (HAV) IRES is thought to be an exception to this rule because it requires intact eIF4F complex for translation. In line with previous results we report that poliovirus (PV) 2A^pro strongly blocks protein synthesis directed by HAV IRES. However, in contrast to previous findings we now demonstrate that eIF4G cleavage by foot-and-mouth disease virus (FMDV) L^pro strongly stimulates HAV IRES-driven translation. Thus, this is the first observation that 2A^pro and L^pro exhibit opposite effects to what was previously thought to be the case in HAV IRES. This effect has been observed both in hamster BHK and human hepatoma Huh7 cells. In addition, this stimulation of translation is also observed in cell free systems after addition of purified L^pro. Notably, in presence of this FMDV protease, translation directed by HAV IRES takes place when eIF2α has been inactivated by phosphorylation. Our present findings clearly demonstrate that protein synthesis directed by HAV IRES can occur when eIF4G has been cleaved and after inactivation of eIF2. Therefore, translation directed by HAV IRES without intact eIF4G and active eIF2 is similar to that observed with other picornavirus IRESs.     

Modulation of eukaryotic mRNA stability via the cap-binding translation complex eIF4F

Decapping by Dcp1 in Saccharomyces cerevisiae is a key step in mRNA degradation. However, the cap also binds the eukaryotic initiation factor (eIF) complex 4F and its associated proteins. Characterisation of the relationship between decapping and interactions involving eIF4F is an essential step towards understanding polysome disassembly and mRNA decay. Three types of observation suggest how changes in the functional status of eIF4F modulate mRNA stability in vivo. First, partial disruption of the interaction between eIF4E and eIF4G, caused by mutations in eIF4E or the presence of the yeast 4E-binding protein p20, stabilised mRNAs. The interactions of eIF4G and p20 with eIF4E may therefore act to modulate the decapping process. Since we also show that the in vitro decapping rate is not directly affected by the nature of the body of the mRNA, this suggests that changes in eIF4F structure could play a role in triggering decapping during mRNA decay. Second, these effects were seen in the absence of extreme changes in global translation rates in the cell, and are therefore relevant to normal mRNA turnover. Third, a truncated form of eIF4E (Delta196) had a reduced capacity to inhibit Dcp1-mediated decapping in vitro, yet did not change cellular mRNA half-lives. Thus, the accessibility of the cap to Dcp1 in vivo is not simply controlled by competition with eIF4E, but is subject to switching between molecular states with different levels of access.