The stimulation of À1 PRF activity by a bimolecular pseudoknot mimicking DU177 suggests an enhancement role for the pseudoknot topology in À1 PRF activity, particularly for those pseudoknots possessing a short stem 1 (22, (25) (26) (27) (28) 32) . In these cases, the pseudoknot topology may just serve to hold the structural determinants of À1 PRF activity together within the same molecule. With loops crossing the grooves of stems under the pseudoknot topology, an interlocked helical junction can be efficiently generated by the junction-flanking stem-loop interactions, and thus restrict the rotational flexibility between stems 2 and 1 as proposed in the torsional restraint hypothesis (18) . In addition, the loop 2 to stem 1 adenine stacking interactions described above can be put in place easily. Alternatively, the fact that most À1 PRF competent pseudoknots possess a short loop 1, thus creating strains (40) , argues for a more active role for the loop-closure constraint imposed by a pseudoknot. In fact, the bimolecular pseudoknot construct still maintains one of the two constrained loops seen in DU177, and the intact DU177 indeed has a much higher À1 PRF efficiency than that of the bimolecular pseudoknot. This can explain why removing one loop-closure constraint from DU177 can reduce its À1 PRF efficiency dramatically, and thus underscores the importance of pseudoknot topology in À1 PRF. In agreement with this work, designed RNA-DNA hybrids, with loop-loop interaction between mRNA and in-trans DNA hairpin to mimick rotational restraints imposed by pseudoknot topology, have been shown to stimulate À1 PRF significantly (18) .