Binding of ATP to eukaryotic initiation factor 2. Differential modulation of mRNA-binding activity and GTP-dependent binding of methionyl-tRNAMetf.
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) (ATP gamma S) inhibits far more weakly. Ternary complex formation is inhibited effectively by ATP, dATP, or ADP, but not by AMP and adenosine. Hence, the gamma-phosphate of ATP and its 3'-OH group are not required for inhibition, but the beta-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 [alpha-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-induced 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 less than or equal to 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.
Gonsky
R
R
Department of Molecular Virology, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Lebendiker
M A
MA
Harary
R
R
Banai
Y
Y
Kaempfer
R
R
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D010408:Penicillium chrysogenum / Q000032:analysis; D014176:Protein Biosynthesis; D012330:RNA, Double-Stranded / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D011817:Rabbits
D003838:Deoxyadenine Nucleotides; D015852:Eukaryotic Initiation Factor-2; D046911:Macromolecular Substances; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D012346:RNA, Transfer, Amino Acyl; C026464:tRNA(m)(Met), methionine-; D000266:Adenylyl Imidodiphosphate; D006165:Guanylyl Imidodiphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; C026600:2'-deoxyadenosine triphosphate