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 degrees . 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.

High-efficiency translational bypassing of non-coding nucleotides specified by mRNA structure and nascent peptide

“…3a). The oligonucleotides did not affect translation as such, because the ribosome can efficiently dissolve duplexes of moderate stability 21 . However, bypassing was abolished (Fig.…”

Mechanical Unfolding of the Beet Western Yellow Virus −1 Frameshift Signal

“…The reconstructions reveal that the pseudoknot interacts with the ribosome at the entrance to the mRNA channel, in close association with a likely mammalian 80S helicase (52). Superimposition of our pseudoknot-stalled and control map shows differences between the maps found at this entrance tunnel (25).…”

“…It has been shown that the ribosome itself acts a helicase comprised of ribosomal proteins S3, S4 and S5 which line the mRNA entrance tunnel in the case of prokaryotic ribosomes (25,52). Specific RNA structures, such as the BWYV pseudoknot, resist unwinding giving rise to increased tension in the spacer and subsequent disruption of the codon-anticodon interaction.…”

A loop 2 cytidine-stem 1 minor groove interaction as a positive determinant for pseudoknot-stimulated –1 ribosomal frameshifting

“…To release this tension, the pseudoknot could be unwound or the mRNA can shift by one nucleotide producing a Ϫ1 frameshift. An alternate model suggests that proteins S3, S4, and S5 of the ribosome function as an RNA helicase to unfold mRNA structures (18,19). A stable three-stranded structure such as a pseudoknot would provide a barrier for the helicase, inhibiting the unwinding of the pseudoknot and preventing forward motion of the elongating ribosome in the reference frame.…”