As demonstrated by the observation that disruption of the adenine stacking between loop 2 and stem 1 of DU177 also affects the À1 PRF efficiency of DU177, there may be more than one way to interfere with the helicase activity of a translocating ribosome. The studies on À1 PRF pseudoknots containing short stem 1, such as BWYV-PK and ScYLV-PK, indicated that specific minor-groove base triples involving S1 and L2 are important for À1 PRF activity, and the impact on frameshift efficiency depends on their relative position to the helical junction (27, 28, 30, 31) . Particularly, it has been shown that junction-flanking tertiary interactions between the 3 0 -nucleotide of loop 2 and base pairing in stem 1 can organize an interhelical interaction network that may act as a kinetic barrier during the unfolding of pseudoknot by ribosome (28) . In contrast, the roles of loop 1 and stem 2 in a À1 PRF pseudoknot stimulator are less addressed as both BWYV-PK and ScYLV-PK have short loop 1 and stem 2 (18) . In this work, we reveal that the major-groove The nucleotides that will interfere with base triple formation in the bimolecular construct are typed in gray. Please note that the slippery site and spacer are not shown in the drawing, and a p2luc reporter, with a full-length firefly luciferase generated as the À1 frame product, was used in this experiment. (B) Results of À1 PRF assays of intermolecular major-groove base triple analysis. (C) Results of À1 PRF assays of intermolecular minor-groove base triple analysis. The designated asterisk indicates the estimated molecular ratios between the in-trans RNA (hTR3 0 ss, 171G3 0 ss and 172U3 0 ss) and the mRNA reporter (hTR5 0 hp and hTR5 0 hpL1c), and the translated proteins corresponding to the 0-frame and À1 frame products are also labeled.