Ribosomal components from Escherichia coli 50 S subunits involved in the reconstitution of peptidyltransferase activity.

Incubation of 50 S subunits from Escherichia coli in the presence of 1.5 M LiCl yielded 1 . 5 ~ core particles which contained -20 proteins and were inactive with respect to peptidyltransferase activity. The proteins of the split fraction were isolated and their effects with respect to restoration of activity were tested in reconstitution assays. Purified L16 was able to restore activity, but the L16 effect was dose-dependent. When present in stoichiometric amounts, L16 had no effect, and the L16-induced activity depended on the presence of at least two out of three “helper” proteins, namely L6, L10, and L11. The helper proteins improved the binding of L16 to the 1 . 5 ~ core particles. The dependence on these helper proteins could be weakened by adding excess amounts of L16 &fold or more). Incubation of 50 S subunits in the presence of 4 M LiCl yielded 4 .0~ cores which contained -10 proteins. Partial and total reconstitution experiments using 4 . 0 ~ cores and (23 S + 5 S) RNA, respectively, together with highly purified proteins revealed that L2, L3, (U), L15, L16, L18, L20, L24, and 23 S RNA are essential components for reconstituting peptidyltransferase activity. As shown previously, L20 and L24 are involved in the early assembly but not in the activity itself. Thus, the remaining seven components must include that or those components which form the active center. Furthermore, seven other components (L13, L17, L21, L22, L25, L27, and 5 S RNA) strongly stimulated the reconstitution of this activity. Six proteins showed a weak effect and 10 no effect, indicating that these latter proteins are not involved in peptidyltransferase activity. The importance of 50 S components for the reconstitution of the peptidyltransferase activity is summarized in Table IV.