Peptide‐Bond Formation on the Ribosome

The acceptor substrate activity of peptidyl transferase of both Escherichia coli and rat liver ribosomes has been investigated using 3′‐N‐aminoacyl analogues of puromycin and 5′‐O‐phosphoryl and 5′‐O‐nucleotidyl derivatives of 3′‐N‐glycylpuromycin aminonucleoside. Two simple assay systems were used with washed ribosomes; the fragment reaction with CpApCpCpA‐(Ac‐L‐[3H]Leu) as donor substrate and the reaction with N‐acetyl‐L‐[3H]phenylalanyl‐tRNA as donor substrate in the presence of poly(U). The results can be summarized as follows. The conditions of the fragment reaction impose severe restrictions on the structural requirements for acceptor substrate activity, especially for E. coli ribosomes. Of the 21 puromycin analogues tested, only 3′‐N‐L‐phenylalanyl‐puromycin aminonucleoside and its 5′‐O‐cytidylyl derivative gave high (>50% of that of puromycin) activity with E. coli ribosomes while all other analogues gave essentially negligible (<5% of that of puromycin) activity. However, with rat liver ribosomes, 3′‐N‐L‐phenylalanyl‐puromycin aminonucleoside gave high activity while all other analogues containing a single benzene ring gave low (5‐24% of that of puromycin) to moderate (25–50% of that of puromycin) activity. These observations plus the low activity of 5′‐O‐cytidylyl (3′‐N‐L‐phenylalanyl)‐puromycin aminonucleoside with rat liver ribosomes represent the most marked differences found between the two types of ribosomes. In the N‐acetyl‐L‐[3H]phenylalanyl‐tRNA assay system with E. coli ribosomes, there was low to moderate activity of a number of the more hydrophobic aminoacyl analogues of puromycin which had negligible activity in the fragment reaction. With rat liver ribosomes, there was a general enhancement of activity of aminoacyl analogues above that seen in the fragment reaction. With the phosphoryl and nucleotidyl analogues, however, only 5′‐O‐cytidylyl(3′‐N‐L‐phenylalanyl)‐puromycin aminonucleoside showed high activity with both types of ribosomes and the results were essentially similar to those of the fragment reaction. In general, therefore, the structural requirements for activity of aminoacyl analogues, but not of nucleotidyl analogues, tended to be more stringent in the fragment reaction as compared with the N‐acetyl‐L‐[3H]phenyl‐alanyl‐tRNA assay. It is suggested that the methanol present in the fragment reaction may cause this by disruption of hydrophobic binding at the puromycin binding site.