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

Abstract

Ribosomal protein L16 was digested with Staphylococcus aureus protease V8 and the resulting peptides were separated by reversed-phase high-performance liquid chromatography. One of the fragments, identified by sequence analysis as the N-terminal peptide of L16, was shown to exhibit partial peptide-bond-formation and transesterification activities of peptidyltransferase upon reconstitution with L16-depleted 50 S core particles. However, several proteins enhanced these activities. L15 increased both reactions when added to the reconstitution mixture, suggesting a limited capacity of the L16 peptide to incorporate into 50s core particles. In contrast, the interaction of L11 with the N-terminal peptide stimulated the transesterification reaction but not the peptidebond-forming activity of ribosomes, indicating a different topological domain for these reactions. Also, EF-P, a soluble protein which reconstructs the peptide-bond formation and transesterification reactions on 70 S ribosomes, stimulated both peptidyltransferase activities exhibited by the L16 N-terminal peptide.Central to the study of the mechanism of peptide-bond synthesis is establishing whether the ribosomal proteins function passively by ensuring correct steric alignment of substrates, or whether they are actively involved in the catalytic process. Thermodynamic considerations and kinetic studies on model compounds with appropriately juxtaposed functional groups indicate that an exclusively template role for the ribosome is at least plausible and may also be sufficient to account for the rate of protein synthesis in vivo [l]. 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]. In addition, our previous chemical modification studies have pointed to the involvement of an active histidine residue during peptide bond synthesis [5 ~ 71. Although other ribosomal proteins (e.g. L2 and L4) also contain such potentially active residues [2], the unique histidine of L16 has to date remained a prime candidate for providing, at least in part, the catalytic activity of the peptidebond formation and transesterification processes [8]. In this connection, we report here on the reconstruction of both peptidyltransferase activities of 50s and 70s ribosomes by proteolytic fragments of L16 and study the effect of L6, L11 and L15 and the stimulatory protein EF-P on these reactions. MATERIALS AND METHODS MaterialsPuromycin dihydrochloride ...

Interactions of elongation factor EF‐P with the Escherichia coli ribosome

“…This is consistent with the EF-P protein being a competitive inhibitor of puromycin [34], an antibiotic known to act at the A-site of the ribosome [35]. Reconstitution experiments, with core particles of the 50S subunit lacking specific 50S proteins, revealed that L16 is essential for the action of the EF-P protein [36]. The crystal structure of the ribosome, with bound aminoacyl-tRNA, indicates that L16 occurs at the A-site of the 50S subunit [37].…”

Crystal structure of elongation factor P from Thermus thermophilus HB8

“…EF-P also interacts with domains II and V of the 23S rRNA, i.e., near the peptidyltransferase center (PTC) (6,7). Ribosome reconstitution experiments have shown that the L16 ribosomal protein or its N-terminal 47-residue fragment was required for EF-P-mediated peptide bond synthesis, whereas L11, L15, or L7͞L12 were not (8)(9)(10).…”