Dear Sir. Concerning your issue about the protocol for the measurment of peptidyl transferase activity . The peptidyl transferase activity of the ribosomes was measured by using the scintillation proximity assay for ribosomal kinetics (SPARK) technique (Polacek et al., 2002), which utilizes [3H]-fMet-tRNA as a donor substrate and biotin-puromycin as an acceptor substrate. Ribosomes (0.4 μM) were preincubated with AUG-containing synthetic mRNA (1.3 μM) and the donor substrate [3H]-fMet-tRNA (0.8 μM), and the reaction was initiated by the addition of biotin-puromycin to a final concentration of 1 μM. In a buffer containing 30 mM MgCl2, the overall rate of puromycin reaction on ribosomes containing L27 that lacked as few as three N-terminal amino acids was significantly reduced compared to those containing wt L27. A similar decrease in the reaction rate of the −3 mutant was observed in control experiments carried out with 1 μM unmodified puromycin. These results demonstrate that L27 truncation impairs the function of the peptidyl transferase center and suggest that the main contribution of L27 to peptidyl transferase activity is associated with its three N-terminal amino acids. I think the following below links may help you in your analysis:

https://ac.els-cdn.com/S1097276505016308/1-s2.0-S1097276505016308-main.pdf?_tid=864802a7-5ea5-44ae-8433-a24752219649&acdnat=1537390949_b0ed363ee5c725a693817ceaba1ef163

http://www.pnas.org/content/pnas/96/1/85.full.pdf

http://rnajournal.cshlp.org/content/2/10/1011.full.pdf

Thanks

Dear Isam Eldin Hussein Elgailani,

Thank you very much for your detailed answer, sure it will help me. I will look for the [3H]-fMet-tRNA that I did not find in a first rapid research.

Many thanks for your help,

Best regards,

Maxime.