As base-triple interactions were shown to be important for À1 PRF activity in several pseudoknots (22, (25) (26) (27) (28) 32) , we disrupted the three major-groove triples and the two minor-groove triples by making mutations on loops 1 and 2 of DU177, respectively. As shown in Figure 2B (lanes 7-9), the À1 PRF efficiencies of both mutants (L1-CCC and L2-GU) were reduced dramatically to below 5%, whereas the CCC/GU mutant, with all five base triples impaired, lost its À1 PRF activity to essentially 0%. The A173G mutant was then made to destroy the crucial Hoogsteen AU base pair that helps in anchoring the interhelical triplex. Similar to what was observed in CCC/GU mutant, this single mutation wiped out À1 PRF activity completely ( Figure 2B , lane 10) and is consistent with the involvement of triplex formation in the stimulation of À1 PRF activity of DU177.