The mutant rpl11b alleles promote opposing effects on tRNA binding to the ribosomal A-and P-sites Sucrose gradient analyses were employed to fractionate cycloheximide arrested elongating ribosomes on mRNAs in lysates generated from JD1313 cells expressing wild-type L11B, 51-4A, and 54-7A. In all strains the 60S peak was smaller than that of the 40S fraction which can be attributed to the presence of only a single copy of RPL11B, which has previously been shown to effectively reduce the number of 60S subunits produced by the cell to 60-66% of true wild-type levels while having no visible phenotypic effect on growth (14) . No significant differences were observed among the samples (data not shown). Phenotypic variation in PRF and in the presence of anisomycin and sparsomycin are indicative of altered interactions between the ribosome and tRNAs. P-site tRNA K d values were determined in vitro by binding 2-fold serial dilutions of N-acetylated-[ 14 C]Phe-tRNA to ribosomes until saturation was achieved ( Figure 4A ), and the resulting data were used to determine steady-state single site binding K d values ( Figure 4B ). Wild-type ribosomes bound this P-site substrate with a K d of 72.3 ± 7.9 nM. The 51-4A mutants promoted a slight increase in affinity for P-site substrate (K d = 50.9 ± 11.2 nM), while 54-7A had the opposite effect (K d = 89.3 ± 10.4 nM). Given the physical interaction between the L11 P-site loop and peptidyl-tRNA, it was imperative to determine whether the observed small changes in P-site affinities promoted by the mutants were biochemically significant. To this end, multiple turnover puromycin reactions were performed. In these experiments, puromycin was added to ribosomes pre-incubated with excess P-site substrate, i.e. Ac-[ 14 C]Phe-tRNA Phe , and accumulation of the peptidylpuromycin product was monitored over time. In these reactions, the first round of peptidylpuromycin synthesis is very rapid. Next, in a slow step, the ribosome intrinsically translocates the deacylated tRNA Phe into the E-site (40), followed by the slow diffusion of Ac-[ 14 C]Phe-tRNA Phe into the P-site where it can react with puromycin. Repetition of this cycle results in slow multiple rounds of product synthesis ( Figure 4C ). Assuming that the L11 mutants do not affect either rates of intrinsic translocation or of Ac-[ 14 C]Phe-tRNA Phe diffusion into the P-site, changes in product accumulation, i.e. K obs , should be due to differences in binding affinities for the P-site substrate. Consistent with this model 51-4A promoted 1.46 ± 0.14-fold increased K obs relative to wild-type ribosomes, while 54-7A decreased K obs to Figure 3 . The L11B mutants promote defects in translational fidelity. Isogenic yeast cells expressing either wild-type or mutant forms of L11B were transformed with dual luciferase reporters and control plasmids and rates of translational recoding were determined. All results are graphed as fold wild type. À1 PRF was measured using the yeast L-A virus frameshift signal. +1 PRF was directed by the frameshift signal derived from the Ty1 retrotransposable element. Nonsense suppression denotes the percentage of ribosomes able to suppress an in-frame UAA termination codon positioned between the Renilla and firefly luciferase reporter genes. Missense suppression rates were evaluated by incorporation of an arginine (AGA) near-cognate amino acid instead of a cognate serine (AGC) at the catalytic codon 218 within the firefly luciferase gene. Error bars denote standard error. P-values are indicated above samples showing statistically significant changes. (Figure 4E and F).