Fluorescence Studies on Association of Human Translation Initiation Factor eIF4E with mRNA cap-Analogues

Abstract

Binding of a long series of mono- and dinucleotide analogues of the 7-methylguanosine containing 5′-mRNA-cap to human protein translation initiation factor eIF4E has been investigated by means of fluorescence. A new methodological approach in gathering and analysis of the fluorescence data provided us with very accurate values of the association equilibrium constant K and normalized, maximal quenching of the protein fluorescence ΔFmax, during titration of eIF4E by various cap-analogues. The results confirm participation of at least two conserved tryptophan residues of eIF4E in interaction with 7-methylguanine, as has been described recently for murine eIF4E, complexed with 7-methyl-GDP in crystal (Marcotrigiano et al., 1997, Cell 89, 951), and for yeast eIF4E, complexed with the same ligand in solution (Matsuo et al., 1997, Nature Struct. Biol. 4, 717). On the other hand binding by eIF4E of unmethylated guanine nucleotides and N2,N2,7-trimethylguanine containing nucleotides differ substantially from the way of binding of the regular mRNA-cap. Influence of the structural features of the cap-analogues, especially the type of the second nucleoside in the dinucleotide caps, on their association with eIF4E and biological activities in in vitro protein translation systems has been discussed in light of the known structures of the eIF4E -7- methyl-GDP complexes in crystal and solution.

Structural and Thermodynamic Behavior of Eukaryotic Initiation Factor 4E in Supramolecular Formation with 4E-Binding Protein 1 and mRNA Cap Analogue, Studied by Spectroscopic Methods.

“…This is in contrast to the published data, 12,13) where the binding preference of m 7 GTP over m 7 GpppA/G was observed. The reasons for the discrepancies between these re- sults and ours are not known at present.…”

Expression, purification and characterization of recombinant mouse translation initiation factor eIF4E as a dihydrofolate reductase (DHFR) fusion protein

“…From a set of three independent measurements the average K d was found to be 10 (±3) nM for 7MeGTP, which is nearly identical to the value (9.1 nM) previously reported for eIF4E purified by cap-affinity chromatography [25]. The K d value is almost 2-orders of magnitude lower than that reported by others [26,29,30], The fluorescence quenching measurements were also conducted with 7MeGDP, 7MeGMP and 7BnGMP under similar condition and the corresponding K d values were found to be 45 (±10) nM, 10,000(±500) nM and 800 (± 200) nm, respectively. Similar trends in the K d values for 7MeGDP (49 nM) and 7MeGMP (1240 nM) were reported by Stolarski's and co-workers [25] Although a Kd value determined by fluorescence titration has not been reported for 7BnGMP, a nearly 9-fold greater K d value (7,000 nM) was recently determined for 7BnGMP by a novel mass spectroscopic technique [47].…”

“…However, extensive dialysis may alter the functional integrity of eIF4E [24,25]. K d values for eIF4E with the cap analogue 7MeGTP have been reported over a broad range from 10 −5 to 10 −8 M −1 [22,23,[25][26][27][28][29][30]. Recently, Stolarski and co-workers have shown that even after repeated dialysis or anion exchange chromatography, 60% of the resulting recombinant eIF4E can still be bound to cap-analogues used for elution [25].…”

Remodeling of the pioneer translation initiation complex involves translation and the karyopherin importin β

“…U1 snRNA, which harbors a trimethylated cap in the cytoplasm, did not detectably coimmunoprecipitate with eIF4E (Fig. 6D), as was evident from several observations: (1) Cellular U snRNPs appear to be largely free of sequestering CBPs, based on their quantitative IP using anti-trimethyl G (Bringmann et al 1983); (2) eIF4E binds trimethylated caps 10-fold less efficiently than it binds monomethylated caps (Wieczorek et al 1999); and (3) snurportin binds the trimethylated cap of cytoplasmic U snRNAs (Will and Lü hrmann 2001) so as to mediate together with IMPb snRNA import into nuclei to function in pre-mRNA splicing (Rollenhagen and Pante 2006;Cook et al 2007).…”