Binding of inosine-substituted mRNA to reticulocyte ribosomes and eukaryotic initiation factors 4A and 4B requires ATP.
We examined the hypothesis that initiation of eukaryotic protein synthesis involves ATP-dependent melting of 5'-cap-proximal secondary structure in mRNA by eukaryotic initiation factors 4A and 4B. In reticulocyte lysate depleted of ribonucleoside triphosphates by pretreatment with hexokinase/glucose, initiation complex formation by native reovirus mRNA showed a strict requirement for ATP. The corresponding mRNA synthesized with ITP in place of GTP to minimize secondary structure also required ATP for binding to 40 S ribosomal subunits in complexes characteristic of initiation. In a partial reaction without ribosomes, purified eukaryotic initiation factors 4A and 4B bound and cross-linked to the capped 5'-end of oxidized mRNA. This interaction was ATP-dependent with inosine-substituted or bromouridine-containing reovirus RNAs as observed previously with native mRNA. The results indicate that if initiation involves ATP-dependent denaturation of mRNA, the effect must occur after initiation factor-mediated attachment of mRNA to the 40 S ribosomal subunit.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D039601:Eukaryotic Initiation Factor-4A; D039642:Eukaryotic Initiation Factors; D007288:Inosine; D009691:Nucleic Acid Denaturation; D010448:Peptide Initiation Factors / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*
D039642:Eukaryotic Initiation Factors; D010448:Peptide Initiation Factors; D012333:RNA, Messenger; C031799:eIF-4B; D007288:Inosine; D000255:Adenosine Triphosphate; D039601:Eukaryotic Initiation Factor-4A