Further characterization of the helicase activity of eIF4A. Substrate specificity.
Eukaryotic initiation factor (eIF) 4A is the archetypal member of the DEAD box family of RNA helicases and is proposed to unwind structures in the 5'-untranslated region of mRNA to facilitate binding of the 40 S ribosomal subunit. The helicase activity of eIF4A has been further characterized with respect to substrate specificity and directionality. Results confirm that the initial rate and amplitude of duplex unwinding by eIF4A is dependent on the overall stability, rather than the length or sequence, of the duplex substrate. eIF4A helicase activity is minimally dependent on the length of the single-stranded region adjacent to the double-stranded region of the substrate. Interestingly, eIF4A is able to unwind blunt-ended duplexes. eIF4A helicase activity is also affected by substitution of 2'-OH (RNA) groups with 2'-H (DNA) or 2'-methoxyethyl groups. These observations, taken together with results from competitive inhibition experiments, suggest that eIF4A may interact directly with double-stranded RNA, and recognition of helicase substrates occurs via chemical and/or structural features of the duplex. These results allow for refinement of a previously proposed model for the mechanism of action of eIF4A helicase activity.
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4935, USA.
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D020121:5' Untranslated Regions / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D039601:Eukaryotic Initiation Factor-4A; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020365:RNA Helicases / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D013379:Substrate Specificity
D020121:5' Untranslated Regions; D009838:Oligodeoxyribonucleotides; D009843:Oligoribonucleotides; D010448:Peptide Initiation Factors; D012333:RNA, Messenger; D039601:Eukaryotic Initiation Factor-4A; D004265:DNA Helicases; D020365:RNA Helicases