Formation of frameshift-stimulating RNA pseudoknots is facilitated by remodeling of their folding intermediates.

Programmed -1 ribosomal frameshifting is an essential regulation mechanism of translation in viruses and bacteria. It is stimulated by mRNA structures inside the coding region. As the structure is unfolded repeatedly by consecutive translating ribosomes, whether it can refold properly each time is important in performing its function. By using single-molecule approaches and molecular dynamics simulations, we found that a frameshift-stimulating RNA pseudoknot folds sequentially through its upstream stem S1 and downstream stem S2. In this pathway, S2 folds from the downstream side and tends to be trapped in intermediates. By masking the last few nucleotides to mimic their gradual emergence from translating ribosomes, S2 can be directed to fold from the upstream region. The results show that the intermediates are greatly suppressed, suggesting that mRNA refolding may be modulated by ribosomes. Moreover, masking the first few nucleotides of S1 favors the folding from S2 and yields native pseudoknots, which are stable enough to retrieve the masked nucleotides. We hypothesize that translating ribosomes can remodel an intermediate mRNA structure into a stable conformation, which may in turn stimulate backward slippage of the ribosome. This supports an interactive model of ribosomal frameshifting and gives an insightful account addressing previous experimental observations.