mRNA Helicase Activity of the Ribosome

Abstract Most mRNAs contain secondary structure, yet their codons must be in single-stranded form to be translated. Until now, no helicase activity has been identified which could account for the ability of ribosomes to translate through downstream mRNA secondary structure. Using an oligonucleotide displacement assay, together with a stepwise in vitro translation system made up of purified components, we show that ribosomes are able to disrupt downstream helices, including a perfect 27 base pair helix of predicted T m = 70°. Using helices of different lengths and registers, the helicase active site can be localized to the middle of the downstream tunnel, between the head and shoulder of the 30S subunit. Mutation of residues in proteins S3 and S4 that line the entry to the tunnel impairs helicase activity. We conclude that the ribosome itself is an mRNA helicase and that proteins S3 and S4 may play a role in its processivity.