We further mutated the stem U-A base pair to the C-G base pair for every U-AÁU major-groove triple (mutants 174W, 175W and 176W in Figure 4A ). As shown in Figure  4B , each of these mutants possesses lower À1 PRF activity than that of DU177 ( Figure 4B, lanes 6, 8 and 10) . The frameshift activities range from 49% for 176W to 22 and 14% for 175W and 174W, respectively. Therefore, mutation on the stem base pair of the distal third major-groove triple, 176W, has less impact on À1 PRF activity than those of the two junction-flanking major-groove triples. Finally, mutating the U of C-G Á U triple (174W or 175W) to the C, thus forming a C-G Á C triple (174T or 175T), can restore frameshift efficiency to the level of DU177. Interestingly, the U-A Á U triple, with a weaker U-A base pair in the stem, seems to have higher À1 PRF activity than the C-G Á U base-triple containing a stronger stem C-G base pair. Taken together, these data strongly suggest that the stem-loop interactions in the major-groove triples and their positions relative to the helical junction play important roles in the À1 PRF activity of DU177.