Some specific changes in rRNA structure are particularly telling. The base stack of aa-tRNA C74 with H. marismortui U2590 (E. coli U2555, yeast U2924) is thought to promote the induced fit of peptidyltransfer (6) . The enhanced deprotection of this base in R247A mutant ribosomes when both A-and P-sites were occupied by tRNAs is consistent with its strong effect on peptidyltransferase activity by this mutation (Figure 3 H) . Interestingly, this base was also strongly deprotected under the same conditions in K237A mutant ribosomes, but in this case peptidyltransferase activity as monitored using the puromycin reaction was actually enhanced ( Figure 3H ). The rates of peptidyltransfer Figure 4 . Continued arrowheads, and those deprotected relative to wild-type are indicated by black arrowheads. Bases marked in gray (A2926 and A2971) were deprotected when the A-site is unoccupied relative to when it contains aa-tRNA. (B) rRNA protection patterns of the L3 basic thumb mutants mapped onto the 2D diagram of 25S rRNA. Arrowheads indicate relatively protected and deprotected bases as above. Colored boxes indicate bases that interact with specified L3 basic thumb amino acid side chains. A2926 and A2971 are circled in gray, and C2925, which is the first gate in the aa-tRNA accommodation corridor, is circled in purple. The three bases participating the Type II A-minor motif that stabilizes the PTC are boxed and indicated. (C) Data from panels A and B mapped onto the 3D structure of the yeast ribosome. Indicated bases colored black correspond to bases deprotected in the mutants, while those colored gray are hyperprotected. Bases participating in the Type II A-minor motif (A m ) are colored purple. Helical structures and the PTC are color coded as indicated. Note that the loop formed between H61-H64 was removed from this figure because it obscures the L3 basic thumb. Table 2 . Homologous yeast 25S rRNA and E. coli 23S rRNA bases pertinent to this study observed in the current study ($0.3 min À1 ) and in similar analyses using E. coli ribosomes are significantly lower than naturally occurring rates [estimated to be >300 s À1 , (58) ] because Ac-Phe-tRNA is a poor substrate for this reaction. However, this property actually enables us to tease out the effects of local structural changes on PTC activity. Recent molecular dynamics simulations of portions of the ribosome reveal that individual bases can undergo a conservable degree of structural mobility due to local Brownian movements [reviewed in (59) ], suggesting that specific bases in the PTC are relatively free to assume either the induced or uninduced conformations in the absence of tRNAs, and that the equilibrium between these two states is influenced by the presence or absence of aa-tRNA in the A-site. Thus, we suggest that R247A mutant drives this equilibrium toward the uninduced conformation, while the K237A mutant favors the induced arrangement. The observation of distinctly different patterns of rRNA protection/ deprotection (e.g. compare G2912, C2929, U2953, G2977, C2985, A2995, G3003, A3006, G3009, U3019, U3023 and A3033 between the K237A and R247A mutants in Figure 4A ) is consistent with the idea that they also have opposing effects on PTC conformation and functionality. In addition, early studies demonstrated that while empty ribosomes are heterogeneous in their affinity for eEF2, consisting of two sub-populations having K d 's for eEF2 ranging from subnanomolar to hundreds of nanomoles (60), the affinity for eEF2 strongly depends on the functional status of the ribosome as determined by the occupancy status of the A-and P-sites (61, 62) . This suggests that the R247A and the double mutants that increased eEF2 affinity shift this equilibrium as well, possibly stabilizing ribosomes in the pre-translocation state, and that the interactions between the 25S rRNA bases and L3 amino acid residues investigated here are involved in transitions between the pre-and post-translocational states.