Binding of ATP and messenger RNA by the beta-subunit of eukaryotic initiation factor 2.
In addition to forming a ternary complex with Met-tRNA(f) and GTP, eukaryotic initiation factor 2 (eIF-2) recognizes a specific site in mRNA molecules. Both binding activities are regulated by ATP, which itself binds tightly and specifically to eIF-2. Denaturation of eIF-2 with urea leads to complete loss of Met-tRNA(f) binding activity, while mRNA binding activity is stable. Hence, distinct conformational features in eIF-2 are required for ternary complex formation and for binding of mRNA. Chromatography of eIF-2 over ATP-agarose, in denaturing conditions that induce polypeptide subunit dissociation, results in selective retention of the beta-subunit of eIF-2. Isolated beta-subunit is capable of binding mRNA as well as ATP. Cibacron blue 3G-A binds tightly to eIF-2 and inhibits the binding of mRNA. This inhibition is relieved upon addition of ATP, showing that Cibacron blue 3G-A competes with ATP for eIF-2. eIF-2 beta subunit, active in binding of mRNA, is recovered upon chromatography of eIF-2 in denaturing conditions over matrix-bound Cibacron blue 3G-A. These results show that the ability of eIF-2 to bind mRNA and its ability to bind ATP are both lodged within remarkably stable domains of its beta-subunit. During initiation of protein synthesis, the eIF-2 beta subunit may thus interact with three ligands important for translational control: Met-tRNA(f), mRNA and ATP.
Gonsky
R
R
Department of Molecular Virology, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Itamar
D
D
Harary
R
R
Kaempfer
R
R
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D012358:RNA, Transfer, Met / Q000378:metabolism*; D014227:Triazines / Q000494:pharmacology; D014508:Urea
D015852:Eukaryotic Initiation Factor-2; D012333:RNA, Messenger; D012358:RNA, Transfer, Met; D014227:Triazines; C012842:Cibacron Blue F 3GA; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D014508:Urea