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 s ubunit. The helicase activity of eIF4A has been further characterized with respect to substrate specificity and directionality. Results confirm that t he initial rate and amplitude of duplex unwinding by eIF4A is dependent on the overall stability, rather than the length or sequence, of the duplex su bstrate. eIF4A helicase activity is minimally dependent on the length of th e single-stranded region adjacent to the double-stranded region of the subs trate. Interestingly, eIF4A is able to unwind blunt-ended duplexes. eIF4A h elicase activity is also affected by substitution of 2 ' -OH (RNA) groups w ith 2 ' -H (DNA) or 2 ' -methoxyethyl groups. These observations, taken tog ether with results from competitive inhibition experiments, suggest that eI F4A may interact directly with double-stranded RNA, and recognition of heli case substrates occurs via chemical and/or structural features of the duple x. These results allow for refinement of a previously proposed model for th e mechanism of action of eIF4A helicase activity.