To assess the effects of the viable mutants on rRNA structure, SHAPE [Selective 2 0 -Hydroxyl Acylation and Primer Extension, (34, 49) ] using 1M7 [1-methyl-7nitroisatoic anhydride, (50) ] was used to probe wild-type and selected mutant ribosomes containing either Ac-aa-tRNA Phe at the P-site alone, or both tRNA Phe and aa-tRNA Phe at the P-and A-sites respectively (Figure 4) . Inspection of the results revealed two general trends. First, that the mutants promoted significant changes in rRNA structure in the peptidyltransferase center (U2953, U2955, G2977), along the path taken by aa-tRNA as it accommodates into the LSU (accommodation corridor, G2912, U2924, C2929, A2934), in Helix 94 where it interacts with the globular domain of L3 (G3003, A3006, G3009), and in Helix 95 (U3019, U3023, A3033). Secondly, while the mutants only altered the Helix 73-Helix 95 region when only the P-site was occupied by Ac-aa-tRNA, the majority of changes in the Helix 91-93 region were observed when both A-and P-sites were occupied. Detailed analyses reveal that the R247A mutant conferred the largest number of changes in rRNA structure, promoting deprotection of C2985 in Helix 91, G3003, A3006, G3009 in Helix 94, and U3019, U3023 and A3033 in Helix 95 when only the P-site was occupied ( Figure 4A and B). In contrast, this mutant promoted increased deprotection of G2912 in Helix 92, U2924 in the A-loop, A2934 in the bulge between Helix 92 and Helix 90, and U2955 in the peptidyltransferase center when the A-site was occupied by aa-tRNA. This mutant also caused hyperprotection of A2987 in Helix 73 when the A-site was occupied. Some of the other mutants had similar effects on some but not all of the same bases, e.g. K237A caused deprotection of G2912 and U2955, and enhanced protection of A2987 when the A-site was occupied. Other mutant specific effects were observed. For example, when only the P-site was occupied K247A promoted enhanced protection of G2977, while both K247A and K236A promoted deprotection of A2995 and U2953 under these conditions ( Figure 4A and B). K237A also promoted increased protection from 1M7 at G2977 when both the A-and P-sites were occupied. R232A had significant effects in the 3 0 loop between H90 and H92, and in the peptidyltransferase center (U2955). The observation that the bulged A2971 in Helix 93 was generally deprotected when only the P-site was occupied by Ac-aa-tRNA Phe , but became protected from chemical attack upon loading of aa-tRNA Phe into the A-site (Figure 4 ) serves as an important control, as this site occupancy-specific conformational change has also been observed for bacterial ribosomes (51) . Interestingly, this pattern was also observed for A2926 in the A-loop, the possible significance of which is discussed below.