The simultaneous slippage model of À1 PRF requires that both the aa-tRNA in the ribosomal A-site, and the peptidyl-tRNA in the P-site must shift on the mRNA (42), while in Ty1 mediated +1 PRF, only the peptidyl-tRNA slips (43) . Consistent with the frameshifting data, all of the L3 basic thumb mutants promoted decreased affinity for aa-tRNA to the A-site ( Figure 3A and B, and summarized in Table 1 ), but did not affect binding of Ac-aa-tRNA to the P-site ( Figure 3C and D, summarized in Table 1 ). Specifically, R232A, which does not directly contact any rRNA bases, had the smallest effect on aa-tRNA binding (K d values $126 nM compared to $94 nM for wild-type, i.e. 1.4-fold increase), while the K236A and K237A mutants, which participate in only a few rRNA contacts ( Figure 4B ), had moderate effects ($165 nM each, 1.8-fold wild-type). In contrast, R247A, which contacts both H61 and H90 had a very strong effect on aa-tRNA binding ($300 nM, 3.3-fold wild-type). The double mutants, which also affected multiple rRNA contacts had comparable effects on aa-tRNA binding (from $245 nM to $340 nM).