In all areas examined, rpl11b ribosomes Y52Q and Y52F matched the wild-type rRNA base modification profile (data not shown), while 51-4A and 54-7A ribosomes revealed consistently reproducible differences. The most significant changes in rRNA structure were observed in bases C2675-A2679 (E. coli numbering: C2306-2310) located in the terminal loop of 25S rRNA H84 ( Figure 5A and E). The two mutants promoted opposing patterns of base protection/deprotection in this structure. Specifically, as compared to wild-type ribosomes, 51-4A promoted enhanced protection of this loop, while the loop was deprotected in the 54-7A mutants. Analysis of the recent cryo-EM yeast ribosome structure (4) revealed that these H84 loop bases are located within 3 Å of the stretches of amino acids changed to alanines in both the 51-4A and 54-7A mutants ( Figure 5B ). These findings suggested that the two mutants had the effects of displacing the P-site loop into two opposing conformational states: extended toward the P-site (54-57A), or retracted into H84 (51-54A). To test whether these two states are naturally dependent on P-site occupancy, the experiments were repeated with wild-type and mutant ribosomes with or without tRNA Phe in their P-sites. Consistent with this model, addition of tRNA to the P-site of wild-type ribosomes resulted in slightly enhanced protection of the H84 terminal loop bases closest to the P-site loop (A2676-A2679). Interestingly, C2675 showed significant deprotection when the P-site was occupied by tRNA. This base is on the far side of the terminal end of H84 from the P-site loop, suggesting that H84 itself alters its conformation upon tRNA occupancy of the P-site ( Figure 5C ). 51-4A's H84 bases were unchanged between P-site bound and unbound ribosomes, consistent with the P-site loop positioned in the 'retracted' state in this mutant, although small differences in the protection patterns suggest that the P-site loop is in a slightly different orientation in this mutant. In contrast, while 54-7A ribosomes, i.e. the P-site loop 'extended' state, showed deprotection at all bases (C2675-A2679) for both P-site bound and salt washed ribosomes, bases A2676-A2679 were less deprotected when tRNA was in the P-site and C2675 was even more reactive, consistent with the notion that the P-site loop interacts with H84 when peptidyl-tRNA is in the P-site.