interactions (see Figure 4A for mutation scheme). As shown in Figure 4B , a dramatic reduction of À1 PRF activity to $10% can be observed for mutants U100C, U101C, A171G and A172U, whereas mutant U102C possesses a À1 PRF efficiency of 33% ( Figure 4B , lanes 1-5). Therefore, the stem-loop interactions flanking the helical junction, contributed by the first two base triples of both stems, are crucial for efficient The enzymatic cleavage results were resolved in a 20% sequencing gel, with the first and eighth wells as alkaline hydrolysis ladder and control, respectively, whereas the last two wells represent guanine and pyrimidine assignment markers. The cleavage results after RNase T2 and V1 treatments, with increment of RNase concentration, are shown in wells 2-4 and 5-7, respectively. In addition, the assigned residues and the corresponding stem/loop regions are listed on the right and left sides of the gel, respectively. Please note that the residues different from each other are boxed for comparison between both RNAs. Finally, the extent of cleavage for each probe is defined as major or minor cut, and summarized in (B) and (D), with gray rhombuses representing RNase T2 cleavage and filled triangles representing RNase V1 cleavage. They are presented along the predicted secondary structures of both RNAs, with the five mutated loop nucleotides in CCC/GU RNA typed in gray. À1 PRF activity. In contrast, the stem-loop interactions from the distal third major-groove triple (U113-A176ÁU102) are less important. During the progress of this work, an extra major-groove base-triple was proposed in the refined DU177 structure (34) . It locates next to the distal third triple, and its formation will be blocked in the L1-ACA mutant created in Figure 2 . Both DU177 and L1-ACA having similar À1PRF efficiencies (compare lane 1 with lane 6 in Figure 2B ) further support the idea that the contribution of a major-groove triple in À1 PRF activity of DU177 also depends on its relative position to the helical junction.