Translation System Engineering

Ribosomal engineering may help elucidate unexplored synthetic potential of the ribosome by manipulation of the peptidyl-transferase center (PTC). However, mutations of many functionally important nucleotides in the PTC are either lethal or highly deleterious for the cell. Therefore, the designed ribosome needs to be functionally isolated from the cellular translation machinery. One solution to this problem is an orthogonal translation system, where both small and large subunits are excluded from the production of cellular proteins and are only engaged in translation of specific mRNAs. The conceptual solution has been achieved recently by engineering of Ribo-T, the ribosome with tethered subunits. Many details of its functionality and assembly remain unknown which precluded further development of the Ribo-T-based orthogonal translation systems. For the first part of this work, we examined the details of the Ribo-T assembly and functionality. It became clear that the covalent linkage between the Ribo-T subunits impedes its assembly and functionality, restricting the bioengineering capacity of such ribosomes. We also attempted to bypass these limitations by developing the principally new design of a specialized translational system. This is achieved by engineering host cells in which Ribo-T carries out translation of cellular proteins, while dissociable ribosome functions as an orthogonal specialized translation machine. Based on this system, a comprehensive library of mutants carrying alterations at every nucleotide of the PTC was generated and it was used to identify the previously unknown aspects of the ribosome’s ability to respond to specific nascent peptide sequences and small molecular weight cofactors. We also used ribosome engineering as a tool for addressing fundamental question why 5S rRNA has been preserved in the course of evolution as a self-standing molecule. By incorporating circularly permutated 5S into 23S rRNAs we engineered a ribosome lacking free 5S rRNA. We showed that the engineered ribosome displays high translational activity while the assembly of the large ribosomal subunit is disrupted. Our results indicate that the evolutionary conservation of 5S rRNA as an independent molecule has been guided primarily by its role in the ribosome assembly, rather than dictated by functional requirements during protein synthesis.