Frameshifting Efficiency is not Determined by the Mechanical Stability of RNA Pseudoknots

Abstract

Ribosomes translate messenger RNA (mRNA) in 3-nucleotide steps, maintaining an open reading frame until a stop codon is reached. Programmed −1 translational frameshifting, whereby the ribosome is forced backward by 1 nt to shift the reading frame, occurs in many viruses. The resulting expression of two proteins from a single mRNA at a fixed ratio is essential for viral propagation. Frameshifting depends on two mRNA structures: a slippery sequence and a downstream pseudoknot. Recent work suggests frameshifting pseudoknots provide greater barriers to unfolding by the ribosomal helicase (1), likely a function of their increased mechanical stability. The mechanical stability of a panel of 8 pseudoknots, associated with different frameshifting efficiencies, was investigated by repeatedly unfolding and refolding single pseudoknot molecules held under tension by optical tweezers. Pseudoknot unfolding was characterized by measuring the associated molecular contour length changes, unfolding forces, and force-dependent kinetics. Most of the pseudoknots we examined unfold at high force (∼30-50 pN). The corresponding contour length changes were consistent with native pseudoknot unfolding. Most often the unfolding occurred in a single step, but for some pseudoknots the dominant unfolding pathway occurred via a partially-folded intermediate at lower force (∼15-20 pN). Surprisingly, we found no obvious correlation between frameshifting efficiency and pseudoknot mechanical stability, indicating that some other property must determine the frameshifting efficiency. These results extend our understanding of the mechanics of RNA pseudoknot structure formation, and the relation between pseudoknot folding and programmed translational frameshifting. (1) Namy, O., Moran, S.J., Stuart, D.I., Gilbert, R.J. & Brierley, I. A mechanical explanation of RNA pseudoknot function in programmed ribosomal frameshifting. Nature 441, 244-247 (2006).