Comprehensive genetic selection revealed essential bases in the peptidyl-transferase center

Abstract

During protein synthesis, the ribosome catalyzes peptide-bond formation. Biochemical and structural studies revealed that conserved nucleotides in the peptidyl-transferase center (PTC) and its proximity may play a key role in peptide-bond formation; the exact mechanism involved remains unclear. To more precisely define the functional importance of the highly conserved residues, we used a systematic genetic method, which we named SSER (systematic selection of functional sequences by enforced replacement), that allowed us to identify essential nucleotides for ribosomal function from randomized rRNA libraries in Escherichia coli cells. These libraries were constructed by complete randomization of the critical regions in and around the PTC. The selected variants contained natural rRNA sequences from other organisms and organelles as well as unnatural functional sequences; hence providing insights into the functional roles played by these essential bases and suggesting how the universal catalytic mechanism of peptide-bond formation could evolve in all living organisms. Our results highlight essential bases and interactions, which are shaping the PTC architecture and guiding the motions of the tRNA terminus from the A to the P site, found to be crucial not only for the formation of the peptide bond but also for nascent chain elongation.