Stimulation of peptidyltransferase reactions by a soluble protein

Abstract

The requirements for peptide‐bond synthesis and transesterification reactions of Escherichia coli 70S ribosomes, 50S native or reconstructed 50S subunits were examined using fMet‐tRNA as donor substrate and puromycin or α‐hydroxypuromycin as acceptors. We report that the soluble protein EF‐P, purified to apparent homogeneity, stimulates the synthesis of N‐formylmethionylpuromycin or N‐formylmethionylhydroxypuromycin by 70S ribosomes or reassociated 30S and 50S subunits. In the presence of EF‐P, 70S ribosomes are significantly more efficient than 50S particles in catalysing either peptide‐bond synthesis or transesterification. The involvement of 50S subunit proteins in EF‐P‐stimulated peptide‐bond formation and transesterification was studied. 50S subunits were dissociated by 2.0 M LiCl into core particles and ‘split' proteins, several of which were purified to homogeneity. When added to 30S·A‐U‐G·f[35S]Met‐tRNA, 50S cores or 50S cores reconstituted with L6 or L11 promoted peptide‐bond synthesis or transesterification poorly. EF‐P stimulated peptide‐bond synthesis by both these types of core particles to approximately the same extent. On the other hand, EF‐P stimulated a low level of transesterification by cores reconstituted with L6 and L11. In contrast, core particles reconstituted with L16 exhibited both peptide‐bond‐forming and transesterification activities and EF‐P stimulated both reactions twentyfold and fortyfold respectively. Thus different proteins differentially stimulate the intrinsic or EF‐P‐stimulated peptide‐bond and transesterification reactions of the peptidyl transferase. Ethoxyformylation of either 50S subunits or purified L16 used to reconstitute core particles, resulted in loss of peptide‐bond formation and transesterification. Similarly ethoxyformylation of EF‐P resulted in a 25–50% loss of its ability to stimulate both reactions. 30S subunits were resistant to treatment by this reagent. These results suggest the involvement of histidine residues in peptidyltransferase activities. The role of EF‐P in the catalytic mechanism of peptidyltransferase is discussed. Copyright © 1985, Wiley Blackwell. All rights reserved