Recognition of Messenger RNA in Eukaryotic Protein Synthesis: Equilibrium Studies of the Interaction between Messenger RNA and the Initiation Factor that Binds Methionyl‐tRNAf

Raymond KAEMPFER*, Rivka HOLLENDER, Hermona SOREQ, Uri NUDEL

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

25 Scopus citations

Abstract

Equilibrium studies of the interaction between messenger RNA and the rabbit reticulocyte initiation factor that binds methionyl‐tRNAf, eIF‐2, were performed, using filtration through nitrocellulose membranes to measure complex formation. The formation of complexes between the initiation factor and messenger RNA is shown to be consistent with a simple, bimolecular equilibrium. Binding of native, translatable 125I‐labeled globin messenger RNA to the factor is tight, with an apparent dissociation constant of approximately 5 × 10 −10 M at physiological salt concentration. This binding does not involve the poly(A) sequence at the 3′ end of globin messenger RNA, nor the 90‐nucleotide sequence adjacent to it, for poly(A)‐free derivatives of globin messenger RNA prepared by controlled, processive phosphorolysis bind the initiation factor with an affinity essentially equal to that of native globin messenger RNA. In contrast to other cases of protein‐nucleic‐acid interaction, binding of initiation factor eIF‐2 to messenger RNA is as tight at 0.15 M KCI as it is at 0.01 M KCI and is consistent with a preference for the RNA conformation existing at physiological salt concentration. 32P‐labeled bacteriophage R17 RNA, a messenger RNA that can be translated, although at relatively low efficiency, in a mammalian cell‐free system, also forms an equimolar complex with eIF‐2, but binds 10 to 15‐fold less tightly than globin messenger RNA. These results show that the eukaryotic initiation factor that binds methionyl‐tRNAf recognizes a sequence in globin messenger RNA that does not include the poly(A) moiety or most of the untranslated sequence at the 3′ end of the molecule. The data suggest a relationship between the affinity of a given messenger RNA species for initiation factor eIF‐2 and its efficiency as a template in protein synthesis.

Original languageEnglish
Pages (from-to)591-600
Number of pages10
JournalEuropean Journal of Biochemistry
Volume94
Issue number2
DOIs
StatePublished - Mar 1979

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