RNA unwinding in translation: assembly of helicase complex intermediates comprising eukaryotic initiation factors eIF-4F and eIF-4B.
Ribosome binding to mRNA requires the concerted action of three initiation factors, eIF-4A, eIF-4B, and eIF-4F, and the hydrolysis of ATP in a mechanism that is not well understood. Several lines of evidence support a model by which these factors bind to the 5' end of mRNA and unwind proximal secondary structure, thus allowing 40S ribosomal subunits to bind. We have previously used an unwinding assay to demonstrate that eIF-4A or eIF-4F in combination with eIF-4B functions as an RNA helicase. To elucidate the molecular mechanism of RNA unwinding, we used a mobility shift electrophoresis assay which allows the simultaneous analysis of unwinding and complex formation between these factors and RNA. eIF-4F forms a stable complex (complex A) with duplex RNA in the absence of ATP. Addition of eIF-4B results in the formation of a second complex (complex B) of slower mobility in the gel. In the presence of ATP, both complexes dissociate, concomitant with the unwinding of the duplex RNA. We present evidence to suggest that unwinding occurs in a processive as opposed to distributive manner. Thus, we conclude that helicase complexes that are formed in the absence of ATP on duplex RNA translocate processively along the RNA in an ATP-dependent reaction and melt secondary structure. These helicase complexes therefore represent intermediates in the unwinding process of mRNA that could precede ribosome binding.
Jaramillo
M
M
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Dever
T E
TE
Merrick
W C
WC
Sonenberg
N
N
D001483:Base Sequence; D004247:DNA; D039562:Eukaryotic Initiation Factor-4F; D039642:Eukaryotic Initiation Factors; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010448:Peptide Initiation Factors / Q000378:metabolism*; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism*; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*
D039562:Eukaryotic Initiation Factor-4F; D039642:Eukaryotic Initiation Factors; D010448:Peptide Initiation Factors; C031799:eIF-4B; D012313:RNA; D004247:DNA; D012316:RNA Nucleotidyltransferases; D020365:RNA Helicases