A comparison between DU177 and several representative À1 PRF pseudoknot stimulators ( Figure 1A -F) revealed a consensus AACAA sequence in the loop 2 of three short stem 1-containing pseudoknots ( Figure 1C -E). Furthermore, both PEMV-1 PK and DU177 contain an unusual Hoogsteen AU base pair, formed between loops 1 and 2, to bridge the helical junction. We thus examined if DU177 could function as a À1 PRF stimulator when it is positioned downstream of a slippery sequence. To this end, the À1 PRF activity of DU177 was measured and compared with those of several well-characterized pseudoknot stimulators available in our laboratory. The results, shown in Figure 1G , indicate that DU177 can act as an efficient À1 PRF stimulator in vitro. The À1 PRF efficiency stimulated by DU177 was comparable with that of the minimal IBV pseudoknot (IBVm-PK) and was in the range of 50%. This value was higher than those of MMTV-PK (24%) and the other two AACAA-containing pseudoknots, BWYV-PK and SRV-PK (21% and 34%, respectively). In addition, DU177 possessed substantial À1 PRF activity in vivo (data not shown). Therefore, the high À1 PRF activity and the well-resolved structure of DU177 (33) . The nucleotides in DU177 are numbered according to those in ref. (33) . The common AACAA sequences are highlighted by gray shadow, the unusual Hoogsteen AU base pairs are boxed, and the adenines stacking and tertiary interactions are represented by filled circles and dotted lines, respectively. (G) The 12% SDS-PAGE analysis of the À1 PRF assays of several viral RNA pseudoknots, including a minimum IBV-PK (IBV m -PK), MMTV-PK, SRV-PK, BWYV-PK and DU177 RNA (22, 25, 29, 32, 33) . Each pseudoknot was incorporated into a pRL-SV40-based reporter, assayed as described in the 'Materials and Methods', and the translated proteins corresponding to the 0-frame and À1 frame products were labeled as indicated. Please note that a negative control without insertion of a pseudoknot only showed background À1 PRF activity (data not shown).