A triplex formed intermolecularly to mimic triplex structure in DU177 can stimulate Z1 PRF Previous studies have shown that the tertiary interactions in human telomerase RNA pseudoknot region can be maintained in a two-piece assembly (35) . Indeed, UV-melting experiments on a bimolecular pseudoknot have indicated that the triplex formation is independent of loop closure embedded in the pseudoknot topology (33) . Because of the dominant role of triplex formation in the À1 PRF activity of DU177 revealed above, we used a similar bimolecular pesudoknot approach to examine the contribution of pseudoknot topology to the À1 PRF activity of DU177. In this approach, the 5 0 -hairpin portion of DU177 was used to replace the DU177 pseudoknot in the reporter mRNA to form the hTR-5 0 hp construct, and an in-trans hTR3 0 ss RNA corresponding to the loop 2 and the 3 0 portion of stem 2 in DU177 was constructed separately ( Figure 5A ). Based on the results from previous UV-melting experiments (33) , the association of hTR3 0 ss RNA with hTR-5 0 hp construct will restore stem 2 and all the triple interactions corresponding to those in the DU177. The bimolecular pseudoknot construct will thus resemble the unimolecular DU177, except for it missing the covalent linkage that connects stem 1 and loop 2. The results shown in Figure 5B indicate that, upon the addition of hTR3 0 ss RNA, the À1 PRF efficiency of 5 0 -hairpin-containing mRNA reporter (hTR-5 0 hp) changes from 0.25 to 5% in a dosage-dependent way. In contrast, a control mRNA reporter, hTR5 0 hp-L1c, designed to disrupt a potential intermolecular major-groove base-triple interactions, does not respond to even a higher dosage of hTR3 0 ss RNA. Similarly, in-trans oligo-nucleotides (171G3 0 ss and 172U3 0 ss RNA), designed to disrupt potential intermolecular minor-groove base-triple interactions, cannot induce significant À1 PRF activity on hTR5 0 hp mRNA reporter, either ( Figure 5C ). Finally, we also examined if an intermolecular U-A Á U major-groove base-triple interaction can be replaced by an intermolecular C-G Á C triple. As shown in Figure 6 , 174T3 0 ss RNA, designed to form a C-G Á C triple with 174T5 0 hp mRNA reporter, can stimulate significant À1 PRF activity on 174T5 0 hp reporter only, but not on hTR5 0 hp or hTR5 0 hp-L1c reporter. Therefore, an intermolecular triplex mimicking the triplex structure spanning the helical junction of DU177, although it does not possess a completed pseudoknot topology, can stimulate À1 PRF activity. Together with the observation that the removal of the continuing triplex structure converts DU177 into a À1 PRF incompetent pseudoknot, these experimental results argue that both the pseudoknot topology and the triplex structure are required for high-efficiency of À1 PRF, with the pseudoknot topology playing an enhancer-like role to further increase À1 PRF efficiency.