A28. A28 is a key nucleotide in stabilization of the pseudoknot fold by anchoring L2 to the S1/S2 junction through formation of two hydrogen bonds with the 2 0 -hydroxyl group of G14. Replacement by U (SF108) or G (SF210) resulted in almost 2-fold decrease whereas a C (SF109), though having a similarly positioned N1 and amino groups as an adenosine, resulted in $4-fold drop in frameshifting ( Figure 1C) . Apparently, the relatively small pyrimidine ring is too far away to form interactions with the 2 0 OH of G14. In addition, the lower frameshift activity of the C-mutant could also be caused by formation of a base pair with G14, which could change the overall structure around the S1/S2 junction. In summary, the effect of single-nucleotide mutations in L2 show the functional, critical importance of the A26:G4-C16 and A28:C5-G15 triples that were previously identified by NMR spectroscopy (26) . Base triples at these positions, stabilizing the S1/S2 junction have been recognized to be essential for frameshifting in other pseudoknots as well, be it with different hydrogen bond patterns (21, 24, 25, 29) . To provide additional evidence for the importance of the specific interactions between L2 and S1 in frameshifting, we accumulated a number of the adverse mutations into one construct. Therefore, a construct was designed with a pyrimidine-rich L2 sequence CUCUCGUG (SF229, 'L2-mut') that included the A21U, C24U, A26G and A28G mutations. SF229 showed an 8-fold lower activity in frameshifting (Table 1) , which is approximately the sum of the single-nucleotide changes.