GTP hydrolysis during methionyl-tRNAf binding to 40 S ribosomal subunits and the site of edeine inhibition.
Three lines of evidence are presented indicating that GTP hydrolysis associated with eukaryotic peptide initiation occurs in the absence of 60 S subunits when methionyl-tRNAf is bound to 40 S ribosomal subunits. An enzyme fraction required for binding of methionyl-tRNAf to 40 S subunits and peptide initiation, tentatively equated with eIF-(4 + 5), has GTPase activity and appears to be responsible for hydrolysis of GTP in the methionyl-tRNAf.eIF-2.GTP complex. Direct analysis of the methionyl-tRNAf.40 S complex formed with with eIF-2 and [8-3H] guanine, [gamma-32P]GTP reveals bound guanine but not gamma-phosphate. Edeine, a peptide antibiotic containing spermidine and beta-tyrosine residues at its COOH terminus and NH2 terminus, respectively, blocks peptide initiation and interferes with binding of methionyl-tRNAf to 40 S ribosomal subunits. Inhibition of binding is observed when the eIF-2-mediated binding reaction is carried out with GTP but not with guanosine 5'-(beta,gamma-methylene)triphosphate or guanosine 5'-(beta,gamma-imido)triphosphate. Edeine was labeled by iodination and shown to bind with high affinity to 40 S but not to 60 S ribosomal subunits. It is suggested that edeine blocks a specific site on the 40 S ribosomal subunit to which a segment of the methionyl-tRNAf molecule is bound during the course of the initiation reaction sequence.
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D004486:Edeine / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D009239:N-Formylmethionine; D010442:Peptide Chain Initiation, Translational / Q000187:drug effects; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
D000900:Anti-Bacterial Agents; D012346:RNA, Transfer, Amino Acyl; D004486:Edeine; D009239:N-Formylmethionine; D006160:Guanosine Triphosphate