Single round assays of peptidyltransferase activity were performed on puromycin treated salt washed ribosomes pre-loaded with Ac-[ 14 C]Phe-tRNA Phe and purified through glycerol cushions (Complex C) as described in the 'Materials and Methods' section. The observed K obs = 0.34 min À1 in wild-type ribosomes ( Figure 3G , Table 1 ) is comparable to similar reactions using E. coli ribosomes and the Ac-Phe-tRNA substrate (44) , confirming that these relatively low rates are determined by Ac-Phe-tRNA as a poor substrate for the peptidyltransferase reaction, and thus represent true measurements of peptidyltransferase activity, as opposed to peptidyl-tRNA turnover or other artifacts. The R232A, K236A and R247A mutants all promoted decreased rates of peptidyltransfer to approximately two-thirds of wild-type levels, while the double mutants (K236A/K237A, K236A/R1247A, and R247A/K248A) had stronger effects, decreasing rates to $50% of wild-type ( Figure 3G and H, Table 1 ). Unexpectedly, the K237A mutant enhanced the rate of peptidyltransfer by almost 2-fold above wild-type levels.