Formation and release of eukaryotic initiation factor 2 X GDP complex during eukaryotic ribosomal polypeptide chain initiation complex formation.
The formation and release of an eukaryotic initiation factor (eIF)-2 X GDP binary complex during eIF-5-mediated assembly of an 80 S ribosomal polypeptide chain initiation complex have been studied by sucrose gradient centrifugation analysis. Isolated 40 S initiation complex reacts with eIF-5 and 60 S ribosomal subunits to form an 80 S ribosomal initiation complex with concomitant hydrolysis of an equimolar amount of bound GTP to GDP and Pi. Sucrose gradient analysis of reaction products revealed that GDP was released from ribosomes as an eIF-2 X GDP complex. Evidence is presented that eIF-5-mediated hydrolysis releases the GTP bound to the 40 S initiation complex as an intact eIF-2 X GDP complex rather than as free GDP and eIF-2 which subsequently recombine to form the binary complex. Furthermore, formation and release of eIF-2 X GDP from the ribosomal complex do not require concomitant formation of an 80 S initiation complex since both reactions occur efficiently when the 40 S initiation complex reacts with eIF-5 in the absence of 60 S ribosomal subunits. These results, along with the observation that the 40 S initiation complex formed with the nonhydrolyzable analogue of GTP, 5'-guanylylmethylene diphosphonate, can neither join a 60 S ribosomal subunit nor releases ribosome-bound eIF-2, suggest that following eIF-5-mediated hydrolysis of GTP bound to the 40 S initiation complex, both Pi and eIF-2 X GDP complex are released from ribosomes prior to the joining of 60 S ribosomal subunits to the 40 S initiation complex.
D000818:Animals; D001156:Artemia / Q000196:embryology / Q000648:ultrastructure; D002417:Cattle; D002499:Centrifugation, Density Gradient; D015852:Eukaryotic Initiation Factor-2; D040061:Eukaryotic Initiation Factor-5; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D010442:Peptide Chain Initiation, Translational; D010448:Peptide Initiation Factors / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012358:RNA, Transfer, Met; D011817:Rabbits; D012270:Ribosomes / Q000378:metabolism*
D015852:Eukaryotic Initiation Factor-2; D040061:Eukaryotic Initiation Factor-5; D006150:Guanine Nucleotides; D010448:Peptide Initiation Factors; D011506:Proteins; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate