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 Met-tRNA as donor substrate and puromycin or a-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 70 S 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 L1 1 . 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.Much of our understanding of the mechanism of protein synthesis and of the topology of ribosomes derives from studies on how these particles catalyze peptide-bond synthesis Several approaches have been used to define the proteins required for the peptidyltransferase activity of ribosomes. These include attempts to localize the individual proteins by affinity labeling of the 3' portion of tRNAs or specific reagents This paper is dedicated to Dr Fritz Lipmann on the occasion of his 85th birthday.Definitions. 1 A230 unit of a protein solution is equivalent to 250 pg [32]. 1.0 AZG0 unit is the quantity ofmaterial contained in 1 ml of a solution which has an absorbance of 1 at 260 mm, when measured in a 1-cm-path-length cell. The concentrations of puromycin and cr-hydroxypuromycin were determined spectrophotometrically in 0.1 M HCl at .2 , , , = 267.5 using E = 2.0 x lo4 M-' cm-' for both compounds.Abbreviations. Abbreviations for nuclei...

Reconstruction of peptidyltransferase activity on 50S and 70S ribosomal particles by peptide fragments of protein L16

“…However, several lines of evidence suggest that the ribosomal peptidyltransferase possesses catalytic residue(s) as well [l, 21. Of the known ribosomal activities, ester aminolysis and ester hydrolysis are most likely to be mediated by some form of catalytic mechanism. In this regard, we have recently reported that ribosomal protein L16 stimulates both the peptide-bond formation and transesterification activities inherent in peptidyltransferase [3]. This study confirmed and extended the findings of previous reconstitution experiments which showed that L16 belongs to a group of 50s subunit components designated as essential to restoring the peptidyltransferase activity of ribosomes [4].…”

“…AUG . f[35S]Met-tRNA complexes and added puromycin or hydroxypuromycin at concentrations of 4 pM and 8 pM, respectively [3].…”

L16, a bifunctional ribosomal protein and the enhancing effect of L6 and L11

“…This bifunctional role was studied by reconstituting L16 into core particles lacking L6, L11 and L16 [30]. Thus, L16 exhibits this role independently of the two stimulatory proteins L6 and L11.…”

(R)-β-Lysine-modified Elongation Factor P Functions in Translation Elongation

“…Further evidence for a role in translation elongation comes from the ability of eIF5A to promote in vitro di-and tripeptide synthesis (12). To date, the effects of EF-P and its ␤-lysine modification on translation have largely been investigated using a puromycin reactivity assay, which does not accurately represent a physiologically relevant peptide synthesis reaction (2,13,14). We now show that EF-P functions in translation elongation, with activity dependent on ␤-lysylation, whereas the additional hydroxyl modification is dispensable.…”