Dominant negative mutants of mammalian translation initiation factor eIF-4A define a critical role for eIF-4F in cap-dependent and cap-independent initiation of translation

EMBO J. 1994 Mar 1;13(5):1205-15.

Abstract

Eukaryotic translation initiation factor-4A (eIF-4A) plays a critical role in binding of eukaryotic mRNAs to ribosomes. It has been biochemically characterized as an RNA-dependent ATPase and RNA helicase and is a prototype for a growing family of putative RNA helicases termed the DEAD box family. It is required for mRNA-ribosome binding both in its free form and as a subunit of the cap binding protein complex, eIF-4F. To gain further understanding into the mechanism of action of eIF-4A in mRNA-ribosome binding, defective eIF-4A mutants were tested for their abilities to function in a dominant negative manner in a rabbit reticulocyte translation system. Several mutants were demonstrated to be potent inhibitors of translation. Addition of mutant eIF-4A to a rabbit reticulocyte translation system strongly inhibited translation of all mRNAs studied including those translated by a cap-independent internal initiation mechanism. Addition of eIF-4A or eIF-4F relieved inhibition of translation, but eIF-4F was six times more effective than eIF-4A, whereas eIF-4B or other translation factors failed to relieve the inhibition. Kinetic experiments demonstrated that mutant eIF-4A is defective in recycling through eIF-4F, thus explaining the dramatic inhibition of translation. Mutant eIF-4A proteins also inhibited eIF-4F-dependent, but not eIF-4A-dependent RNA helicase activity. Taken together these results suggest that eIF-4A functions primarily as a subunit of eIF-4F, and that singular eIF-4A is required to recycle through the complex during translation. Surprisingly, eIF-4F, which binds to the cap structure, appears to be also required for the translation of naturally uncapped mRNAs.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Chloramphenicol O-Acetyltransferase / biosynthesis
  • Chloramphenicol O-Acetyltransferase / metabolism
  • Eukaryotic Initiation Factor-4A
  • Eukaryotic Initiation Factor-4F
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Peptide Chain Initiation, Translational*
  • Peptide Initiation Factors / genetics
  • Peptide Initiation Factors / metabolism*
  • Point Mutation
  • Protein Biosynthesis
  • RNA Caps / metabolism*
  • RNA Helicases
  • RNA Nucleotidyltransferases / metabolism
  • RNA, Messenger / metabolism*
  • Rabbits
  • Reticulocytes / metabolism
  • Ribosomes / metabolism
  • Transcription, Genetic

Substances

  • Eukaryotic Initiation Factor-4F
  • Peptide Initiation Factors
  • RNA Caps
  • RNA, Messenger
  • Chloramphenicol O-Acetyltransferase
  • Eukaryotic Initiation Factor-4A
  • RNA Nucleotidyltransferases
  • RNA Helicases