Amino acid sequence of the mRNA cap-binding protein from human tissues.

Rychlik, Wojciech; Domier, Leslie L.; Gardner, Paul R.; Hellmann, Gary M.; Rhoads, Robert E.

doi:10.1073/pnas.84.4.945

Cited by 110 publications

(64 citation statements)

References 44 publications

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“…eIF4E Expression and Purification-Full-length wild-type human eIF4E (14) was expressed in Escherichia coli BL21 (COE) pLys(S) from pET11d-eIF4E (64). Protein expression was induced with 0.4 mM isopropyl-␤-D-thiogalactopyranoside overnight at 15°C.…”

Section: Methodsmentioning

confidence: 99%

Stopped-flow Kinetic Analysis of eIF4E and Phosphorylated eIF4E Binding to Cap Analogs and Capped Oligoribonucleotides

Slepenkov

Darżynkiewicz

Rhoads

2006

Journal of Biological Chemistry

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Recruitment of eukaryotic mRNA to the 48 S initiation complex is rate-limiting for protein synthesis under normal conditions. Binding of the 5-terminal cap structure of mRNA to eIF4E is a critical event during this process. Mammalian eIF4E is phosphorylated at Ser-209 by Mnk1 and Mnk2 kinases. We investigated the interaction of both eIF4E and phosphorylated eIF4E (eIF4E(P)) with cap analogs and capped oligoribonucleotides by stopped-flow kinetics. For m 7 GpppG, the rate constant of association, k on , was dependent on ionic strength, decreasing progressively up to 350 mM KCl, but the rate constant of dissociation, k off , was independent of ionic strength. Phosphorylation of eIF4E decreased k on by 2.1-2.3-fold at 50 -100 mM KCl but had progressively less effect at higher ionic strengths, being negligible at 350 mM. Contrary to published evidence, eIF4E phosphorylation had no effect on k off . Several observations supported a simple one-step binding mechanism, in contrast to published reports of a two-step mechanism. The kinetic function that best fit the data changed from single-to double-exponential as the eIF4E concentration was increased. However, measuring k off for dissociation of a pre-formed eIF4E⅐m 7 GpppG complex suggested that the double-exponential kinetics were caused by dissociation of eIF4E dimers, not a two-step mechanism. Addition of a 12-nucleotide chain to the cap structure increased affinity at high ionic strength for both eIF4E (24-fold) and eIF4E(P) (7-fold), primarily due to a decrease in k off . This suggests that additional stabilizing interactions between capped oligoribonucleotides and eIF4E, which do not occur with cap analogs alone, act to slow dissociation.The efficiency of mRNA translational initiation is strongly enhanced by the 5Ј-terminal cap, m 7 GpppN (1). The cap specifically binds to eIF4E, 2 which may be the first canonical initiation factors to interact with mRNA during its recruitment to the ribosome. eIF4E in turn binds to eIF4G, a protein that also interacts with the RNA helicase eIF4A to promote unwinding of mRNA secondary structure, with the multisubunit factor eIF3 to recruit the 43 S initiation complex, and with the cytoplasmic poly(A)-binding protein to enhance initiation of poly(A)-containing mRNAs. Initiation codon recognition is followed by dissociation of eIFs and joining of the 60 S ribosomal subunit to form the elongation-competent 80 S initiation complex. eIF4E has been extensively investigated in organisms that range from yeast to mammals (2-7). Besides translation, eIF4E also functions in nucleocytoplasmic transport of mRNA, sequestration of mRNA in a nontranslatable state, and stabilization of mRNA against decay in the cytosol (8 -10). The three-dimensional structures of human, mouse, and Saccharomyces cerevisiae eIF4E have been solved (11-13). The complex of full-length human eIF4E with m Cap analogs bind to eIF4E in a tight complex, a step that has been studied primarily by equilibrium techniques (15-33). Intrinsic Trp fluorescence quenching of N-termi...

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Section: Methodsmentioning

confidence: 99%

Stopped-flow Kinetic Analysis of eIF4E and Phosphorylated eIF4E Binding to Cap Analogs and Capped Oligoribonucleotides

Slepenkov

Darżynkiewicz

Rhoads

2006

Journal of Biological Chemistry

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“…Preparation of unfolded target proteins. Cloned cDNAs encoding the entire coding regions for ,B-actin (11), actinvertebrate actin-related protein (RPV) (26), (x- (47), 3-(48), and y-tubulin (50), TCP-1 (1), cyclin B (36), cap-binding protein (40), c-Myc (37), and p2lras (H-Ras) (25) were expressed as labeled polypeptides in E. coli BL21(DE3) by using pET vectors (43), and the labeled denatured proteins were purified from insoluble inclusion bodies as described before (16,17,31). The radiochemical and biochemical purity of each target protein was determined by analysis on SDS-polyacrylamide gels; this allowed an accurate estimation of specific radioactivity.…”

Section: Methodsmentioning

confidence: 99%

Facilitated folding of actins and tubulins occurs via a nucleotide-dependent interaction between cytoplasmic chaperonin and distinctive folding intermediates.

Melki¹,

Cowan²

1994

Mol. Cell. Biol.

107

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In the cytoplasm of eukaryotes, the folding of actins and tubulins is facilitated via interaction with a heteromeric toroidal complex (cytoplasmic chaperonin). The folding reaction consists of the formation of a binary complex between the unfolded target protein and the chaperonin, followed by the ultimate release of the native polypeptide in an ATP-dependent reaction. Here we show that the mitochondrial chaperonin (cpn6O) and the cytoplasmic chaperonin both recognize a range of target proteins with different relative affinities; however, the cytoplasmic chaperonin shows the highest affinity for intermediates derived from unfolded tubulins and actins. These high-affinity actin and tubulin folding intermediates are distinct from the "molten globule" intermediates formed by noncytoskeletal target proteins in that they form relatively slowly. We show that the interaction between cytoplasmic chaperonin and unfolded target proteins depends on the chaperonin being in its ADP-bound state and that the release of the target protein occurs after a transition of the chaperonin to the ATP-bound state. Our data suggest a model in which ATP hydrolysis acts as a switch between conformational forms of the cytoplasmic chaperonin that interact either strongly or weakly with unfolded substrates.The biological function of most, if not all, proteins depends critically upon their three-dimensional structure. Although the information contained in the linear sequence of amino acids is thought to be sufficient. to specify this structure (2, 24), the correct folding of many proteins is not a spontaneous process under physiological conditions; rather, there is a requirement for interaction with a class of proteins or protein complexes known as molecular chaperones (reviewed in references 14, 18, and 39). In particular, one class of chaperones (typified by GroEL in prokaryotes) consist of multisubunit toroidal ring assemblies and are believed to function by providing a sequestered environment in which aberrant folding and aggregation are prevented; the correctly folded polypeptide is ultimately discharged in an Mg-ATP-dependent reaction. These toroidal structures are termed chaperonins (6,10,19,28,34,35,46). Actin and tubulin are the major soluble cytoplasmic proteins in eukaryotic cells and are the subunits from which actin filaments and microtubules are assembled. The folding of actins, tubulins, and actin-and tubulin-related proteins is facilitated by a chaperonin (15-17, 31, 49) that differs from its prokaryotic and mitochondrial homologs in that it is heteromeric: the multisubunit toroidal structure is assembled from eight different (though related) polypeptides (15, 38), one of which is the t-complex polypeptide TCP-1 (27, 42). As is the case for other chaperonins, the cytoplasmic chaperonin forms a stable binary complex with unfolded target polypeptides in the absence of Mg-ATP, and the hydrolysis of Mg-ATP is absolutely required for the generation of folded proteins (15-17). However, an important difference exists between the mecha...

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“…2. Alignment of the deduced amino acid sequences for Xenopus laevis, human [6], rabbit [5] and mouse [4] elF-4E protein. The italicized sequence is included in the long type cDNA.…”

Section: Rl' Wede Knkrg Grw'lz Tlnkq ~Rnd Ldrfw Letlm Clige Sfdbh 8ddvcmentioning

confidence: 99%

“…The activity of elF-4E is physiologically regulated by phosphorylation at Ser-53 [2]. cDNAs or genes for elF-4E have been cloned from yeast [3], mouse [4], rabbit [5], human [6] and wheat [7,8]. The amino acid sequence as deduced from the cDNA clones predicts the presence of 8 tryptophans (9 in wheat elF-4E) that are evolutionarily remarkably conserved in number and position.…”

Section: Introductionmentioning

confidence: 99%

mRNA encoding the translation initiation factor eIF‐4E is expressed early in Xenopus embryogenesis

et al. 1995

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The translation initiation factor elF-4E plays an important role in regulating the overall rate of translation in eukaryotic cells. To investigate the expression of elF-4E itselF, we characterized the elF-4E mRNA expressed in Xenopus embryos. 5'-RACE was performed to determine the 5'-end of the mRNA and the result predicts isoforms differing at the amino-terminal end. Expression of the elF-4E mRNA in Xenopus oocytes and embryos was examined by RT-PCR. Xenopus elF-4E mRNA is produced during oogenesis and persists during the early stages of embryogenesis as a maternal mRNA.

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Amino acid sequence of the mRNA cap-binding protein from human tissues.

Cited by 110 publications

References 44 publications

Stopped-flow Kinetic Analysis of eIF4E and Phosphorylated eIF4E Binding to Cap Analogs and Capped Oligoribonucleotides

Stopped-flow Kinetic Analysis of eIF4E and Phosphorylated eIF4E Binding to Cap Analogs and Capped Oligoribonucleotides

Facilitated folding of actins and tubulins occurs via a nucleotide-dependent interaction between cytoplasmic chaperonin and distinctive folding intermediates.

mRNA encoding the translation initiation factor eIF‐4E is expressed early in Xenopus embryogenesis

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