TY - JOUR
T1 - Binding of ATP to eukaryotic initiation factor 2
T2 - Differential modulation of mRNA-binding activity and GTP-dependent binding of methionyl-tRNAfMet
AU - Gonsky, Rivkah
AU - Lebendiker, Mario A.
AU - Harary, Ronit
AU - Banai, Yona
AU - Kaempfer, Raymond
PY - 1990/6/5
Y1 - 1990/6/5
N2 - Eukaryotic initiation factor 2 (eIF-2) is shown to bind ATP with high affinity. Binding of ATP to eIF-2 induces loss of the ability to form a ternary complex with Met-tRNAf and GTP, while still allowing, and even stimulating, the binding of mRNA. Ternary complex formation between eIF-2, GTP, and Met-tRNAf is inhibited effectively by ATP, but not by CTP or UTP. Hydrolysis of ATP is not required for inhibition, for adenyl-5′-yl imidodiphosphate (AMP-PNP), a nonhydrolyzable analogue of ATP, is as active an inhibitor; adenosine 5′-O-(thiotriphosphate) (ATPγS) inhibits far more weakly. Ternary complex formation is inhibited effectively by ATP, dATP, or ADP, but not by AMP and adenosine. Hence, the γ-phosphate of ATP and its 3′-OH group are not required for inhibition, but the β-phosphate is indispensible. Specific complex formation between ATP and eIF-2 is shown 1) by effective retention of Met-tRNAf- and mRNA-binding activities on ATP-agarose and by the ability of free ATP, but not GTP, CTP, or UTP, to effect elution of eIF-2 from this substrate; 2) by eIF-2-dependent retention of [α-32P]ATP or dATP on nitrocellulose filters and its inhibition by excess ATP, but not by GTP, CTP, or UTP. Upon elution from ATP-agarose by high salt concentrations, eIF-2 recovers its ability to form a ternary complex with Met-tRNAf and GTP. ATP-in-duced inhibition of ternary complex formation is relieved by excess Met-tRNAf, but not by excess GTP or guanyl-5′-yl imidodiphosphate (GMP-PNP). Thus, ATP does not act by inhibiting binding of GTP to eIF-2. Instead, ATP causes Met-tRNAf in ternary complex to dissociate from eIF-2. Conversely, affinity of eIF-2 for ATP is high in the absence of GTP and Met-tRNAf (Kd ≤ 10-12 M), but decreases greatly in conditions of ternary complex formation. These results support the concept that eIF-2 assumes distinct conformations for ternary complex formation and for binding of mRNA, and that these are affected differently by ATP. Interaction of ATP with an eIF-2 molecule in ternary complex with Met-tRNAf and GTP promotes displacement of Met-tRNAf from eIF-2, inducing a state favorable for binding of mRNA. ATP may thus regulate the dual binding activities of eIF-2 during initiation of translation.
AB - Eukaryotic initiation factor 2 (eIF-2) is shown to bind ATP with high affinity. Binding of ATP to eIF-2 induces loss of the ability to form a ternary complex with Met-tRNAf and GTP, while still allowing, and even stimulating, the binding of mRNA. Ternary complex formation between eIF-2, GTP, and Met-tRNAf is inhibited effectively by ATP, but not by CTP or UTP. Hydrolysis of ATP is not required for inhibition, for adenyl-5′-yl imidodiphosphate (AMP-PNP), a nonhydrolyzable analogue of ATP, is as active an inhibitor; adenosine 5′-O-(thiotriphosphate) (ATPγS) inhibits far more weakly. Ternary complex formation is inhibited effectively by ATP, dATP, or ADP, but not by AMP and adenosine. Hence, the γ-phosphate of ATP and its 3′-OH group are not required for inhibition, but the β-phosphate is indispensible. Specific complex formation between ATP and eIF-2 is shown 1) by effective retention of Met-tRNAf- and mRNA-binding activities on ATP-agarose and by the ability of free ATP, but not GTP, CTP, or UTP, to effect elution of eIF-2 from this substrate; 2) by eIF-2-dependent retention of [α-32P]ATP or dATP on nitrocellulose filters and its inhibition by excess ATP, but not by GTP, CTP, or UTP. Upon elution from ATP-agarose by high salt concentrations, eIF-2 recovers its ability to form a ternary complex with Met-tRNAf and GTP. ATP-in-duced inhibition of ternary complex formation is relieved by excess Met-tRNAf, but not by excess GTP or guanyl-5′-yl imidodiphosphate (GMP-PNP). Thus, ATP does not act by inhibiting binding of GTP to eIF-2. Instead, ATP causes Met-tRNAf in ternary complex to dissociate from eIF-2. Conversely, affinity of eIF-2 for ATP is high in the absence of GTP and Met-tRNAf (Kd ≤ 10-12 M), but decreases greatly in conditions of ternary complex formation. These results support the concept that eIF-2 assumes distinct conformations for ternary complex formation and for binding of mRNA, and that these are affected differently by ATP. Interaction of ATP with an eIF-2 molecule in ternary complex with Met-tRNAf and GTP promotes displacement of Met-tRNAf from eIF-2, inducing a state favorable for binding of mRNA. ATP may thus regulate the dual binding activities of eIF-2 during initiation of translation.
UR - http://www.scopus.com/inward/record.url?scp=0025302825&partnerID=8YFLogxK
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C2 - 2111815
AN - SCOPUS:0025302825
SN - 0021-9258
VL - 265
SP - 9083
EP - 9089
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 16
ER -