Investigating Ribosomal Catalysis: Optimizing Assays with Thermostable Bacterial Ribosomes for Defining Minimal Components Necessary for Peptidyl Transferase Activity

Abstract

Ribosomes are cellular molecular machinery, facilitating the peptidyl transferase activity – a crucial reaction for the elongation of the peptidyl chain during translation. The process of how exactly ribosome catalyzes peptidyl transferase activity remains a subject of study. Researchers are determined to establish the minimal ribosomal components essential for ribosomal activity. Studies with E. coli 70S ribosome showed that only large ribosomal subunit 50S is essential for performing the peptidyl transferase activity. Later, it was discovered that only one of 50S rRNAs, 23S rRNA, is essential, as it contains the peptidyl transferase center. Later studies with more thermophilic bacteria species paved the way to create a minimal synthetic ribosome reconstructed from the in vitro synthesized ribosomal rRNAs. This project aims to create a minimal Thermus thermophilus ribosome from in vitro transcribed (IVT) rRNAs and naturally extracted 50S total protein components and assay its peptidyl transferase activity with fragment reaction to test the functionality of the reconstructed large ribosomal subunit. For this purpose, a new assay with f[35S]Met-tRNAfMet was assembled. The results of this work show that using [35S] radiolabel leads to more sensitive peptidyl transferase activity detection. On the other hand, the detected activity of the reconstructed Thermus thermophilus 50S subunit is still low, which significantly differs from studies with closely related bacteria species. That opens a question of whether post-translational modifications matter for the activity of Thermus thermophilus ribosomes.