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Eukaryotic peptide chain initiation: A study using fluorescent probes
Abstract
The minimum factors required for efficient formation of both the ternary complex (eIF-2$\cdot$GTP$\cdot$Met-tRNA$\sb{\rm f}\sp{\rm Met}$) and the 40S initiation complex (40S$\cdot$message$\cdot$Met-tRNA$\sb{\rm f}\sp{\rm Met}$) have been highly purified and characterized by filtration assays. In our proposed model, eIF-2B first catalyzes GTP/GDP exchange on eIF-2 in physiological Mg$\sp{2+}$ concentrations (1-2 mM). Next, eIF-3 promotes ternary complex formation as well as the formation of 40S complex. Co-eIF-2C, having both eIF-2B and eIF-3 activities, can substitute the latter two factors. Co-eIF-2A also promotes ternary complex formation and inhibits mRNA-induced ternary complex dissociation. Formation of the 40S complex is enhanced when a translatable message is present (either a synthetic AUG-containing polyribonucleotide or globin mRNA). In order to further study protein-protein and protein-RNA interactions, fluorescent derivatives of GDP and ATP were developed for use in steady-state fluorescence spectroscopy experiments. The probes were synthesized by reaction of a periodate oxidized ribonucleotide and a fluorescent amine-containing derivative with subsequent hydride reduction. The structure regarding the modification to the ribose ring has been proposed based UV-vis, NMR and FAB-MS studies. Tryptophan and fluorescein labeled GDP were used as probes to observe conformational changes in eIF-2 in response to Mg$\sp{2+}$ concentration. Fluorescein labeled ATP (ATP*F) has been shown to bind specifically and reversibly to eIF-2 with high affinity. The binding of ATP*F to eIF-2 was independent of GTP binding and was used to measure the binding of p$\sp{67}$ (a key regulatory factor) to eIF-2 (K$\sb{\rm d}\approx$ 1 $\mu$M). ATP*F was also used as a probe to characterize binding of eIF-2 to all the other factors involved in 40S complex formation. Using a combination of both spectroscopic and filtration assays, the binary association constants between quaternary complex (Q), message (M) and 40S ribosomes (R) were measured. The message enhancement observed in 40S complex formation was thermodynamically quantitated and characterized as a cooperative system. A 79-fold difference was calculated for K$\sb{\rm a(Q,R)}$ and K$\sb{\rm a(Q,R\cdot M)}$.
Subject Area
Biochemistry|Organic chemistry
Recommended Citation
Hileman, Ronald E, "Eukaryotic peptide chain initiation: A study using fluorescent probes" (1993). ETD collection for University of Nebraska-Lincoln. AAI9415967.
https://digitalcommons.unl.edu/dissertations/AAI9415967