Probing the function of the eucaryotic 5' cap structure by using a monoclonal antibody directed against cap-binding proteins.
A monoclonal antibody directed against cap-binding proteins was used to elucidate the possible mechanism by which cap-binding proteins function in initiation of eucaryotic translation. The monoclonal antibody preparation employed in this study exhibited a marked differential effect in inhibiting the translation of folded, capped eucaryotic-mRNAs to a far greater extent than naturally uncapped mRNAs or native capped mRNAs that do not possess extensive 5' end secondary structure. These findings were consistent with the effects of the antibody on initiation complex formation with three different types of reovirus mRNA: native reovirus mRNA; inosine-substituted reovirus mRNA, which has a relaxed secondary structure; and bromouridine-substituted reovirus mRNA, in which base pairing is enhanced relative to regular reovirus mRNA. The extent that translation initiation complex formation was inhibited by the monoclonal antibody directly correlated to the degree of secondary structure present in the mRNA. Binding of bromouridine-substituted reovirus mRNA to ribosomes was inhibited to the greatest extent, while binding of inosine-substituted reovirus mRNA was not inhibited at all in the reticulocyte lysate system or was slightly inhibited in a wheat-germ system. These results support the hypothesis that cap-binding proteins are involved in unwinding of the 5' terminal, secondary structure of many eucaryotic mRNAs, thus facilitating the attachment of ribosomes to mRNA.
D000818:Animals; D000911:Antibodies, Monoclonal; D066298:In Vitro Techniques; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D039381:RNA Cap-Binding Proteins; D012315:RNA Caps / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000276:immunology / Q000502:physiology*; D011817:Rabbits
D000911:Antibodies, Monoclonal; D039381:RNA Cap-Binding Proteins; D012315:RNA Caps; D012333:RNA, Messenger; D016601:RNA-Binding Proteins