HISTIDINE-229 IN PROTEIN L2 IS APPARENTLY ESSENTIAL FOR 50S PEPTIDYL TRANSFERASE-ACTIVITY

It has recently been suggested that peptidyl transferase activity is p rimarily a property of ribosomal RNA and that ribosomal proteins may a ct only as scaffolding. On the other hand, evidence from both photoaff inity labeling studies and reconstitution studies suggest that protein L2 may be functionally important for peptidyl transferase. In the wor k reported here, we reconstitute 50S subunits in which the H229Q varia nt of L2 replaces L2, with all other ribosomal components remaining un changed, and determine the catalytic and structural properties of the reconstituted subunits. We observe that mutation of the highly conserv ed His 229 to Gin results in a complete loss of peptidyl transferase a ctivity in the reconstituted 50S subunit. This is strong evidence for the direct involvement of L2 in ribosomal peptidyl transferase activit y. Control experiments show that, though lacking peptidyl transferase activity, 50S subunits reconstituted with H229Q-L2 appear to be identi cal with 50S subunits reconstituted with wild-type L2 with respect to protein composition and 70S formation in the presence of added 30S sub units. Furthermore, as shown by chemical footprinting analysis, H229Q- L2 appears to bind 23S RNA in the same manner as wild-type L2. Thus, t he effect of H229 mutation appears to be confined to an effect on pept idyl transferase activity, providing the most direct evidence for prot ein involvement in this function to date.