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.
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.
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Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Méthot
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N
Svitkin
Y
Y
Merrick
W C
WC
Sonenberg
N
N
D000595:Amino Acid Sequence; D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000378:metabolism; D039601:Eukaryotic Initiation Factor-4A; D039562:Eukaryotic Initiation Factor-4F; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010442:Peptide Chain Initiation, Translational; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D014176:Protein Biosynthesis; D012315:RNA Caps / Q000378:metabolism*; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D014158:Transcription, Genetic
D039562:Eukaryotic Initiation Factor-4F; D010448:Peptide Initiation Factors; D012315:RNA Caps; D012333:RNA, Messenger; D015500:Chloramphenicol O-Acetyltransferase; D039601:Eukaryotic Initiation Factor-4A; D012316:RNA Nucleotidyltransferases; D020365:RNA Helicases