base-triple interactions closer to the helical junction are more important than the distal ones for À1 PRF activity of DU177 ( Figures 2B and 4B) . Therefore, the relative position toward the helical junction is important for both major-and minor-groove base-triple in determining the contribution of a base-triple to the À1 PRF stimulation activity of a pseudoknot. This position-dependent effect can be rationalized by the stabilization of stem 2 by stem-loop interactions, and the anchoring of stem 2 into stem 1 via the junction bridging triplex formation. Such interactions will provide extra restraints during the unwinding of stem 1 by ribosome and are consistent with the torsional restraint hypothesis (18) . Both the helical geometry of a pseudoknot and its ability to against deformation by a ribosome can be affected by the stem-loop interaction in a positiondependent way, too. Indeed, modulation of frameshift efficiency by the modification of junction geometry via tertiary interactions has been observed in the helical junction of MMTV-PK and BWYV-PK (29, 30) . In particular, the DU177 pseudoknot also adopts a bent conformation around the helical junction (33, 34) , and the tertiary stem-loop interactions contributed by the junction-flanking base triples may thus play a unique role to facilitate this bending process. Alternatively, the single-molecule unfolding analysis of a set of DU177 variants, with À1 PRF efficiency ranging from 50 to 0%, reveals a correlation between pseudoknot mechanical stability and frameshift efficiency (38) . Building upon this foundation, the position effect of stem-loop interaction on the mechanical stability of a pseudoknot can be analyzed further in the future.