make it a good model molecule for dissecting the structural determinants within a pseudoknot for the stimulation of À1 PRF. A structural feature found in several À1 PRF pseudoknot stimulators, which involved stacking of the loop 2 adenines in the minor groove of stem 1 in a pseudoknot, was also identified in DU177 (33) . The residues 166-168 of DU177 were thus mutated, and the resulting mutants (C166U, A167C, A168U and L2-UU in Figure 2A ) still possessed substantial À1 PRF activities ranging from 50% to 30% ( Figure 2B, lanes 2-5) . Similarly, mutagenesis on loop 1 nucleotides 103 and 105 generated mutant L1-ACA with a DU177-like À1 PRF activity (compare lane 1 with lane 6 in Figure 2B ). Therefore, the À1 PRF activity of DU177 is not very sensitive to nucleotide identity in the 3 0 portion of its loop 1.