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.nucleotide essentiality ͉ protein biosynthesis ͉ symmetrical region R ibosomes are universally conserved ribonucleoproteins that translate the genetic information contained in mRNAs into proteins. The large (50S) ribosomal subunit catalyzes peptide-bond formation at the peptidyl-transferase center (PTC) between aminoacyl-tRNA (aa-tRNA) bound to the A site and peptidyl-tRNA (pep-tRNA) at the P site. In the crystal structures of 50S subunits of Haloarcula marismortui, H50S (1, 2), Deinococcus radiodurans, D50S (3, 4), and 70S ribosomes (5, 6), the PTC is composed solely of 23S rRNA and, hence, acts as a ribozyme, consistent with biochemical findings using deproteinized 50S subunit (7-10) for the formation of a single peptide bond.The PTC provides the frame for peptide-bond formation (11, 12) and plays a critical role in tRNA and nascent chain release (13-17), and the global ribosomal architecture is crucial for substrate positioning (18,19). Consistently, the hypothesis, based on structures of H50S complexed with minimum substrates, that the PTC acts as a general acid-base catalyst (2, 20-22), was contradicted by various mutagenesis and biochemical studies (12,(23)(24)(25)(26)(27)(28). The main catalytic contribution of the ribosomes, substrates positioning at proper orientation (4,28,29), is achieved by remote interactions, accompanied by symmetrical base-pairing of C75 of both tRNAs with G2553 (Escherichia coli numbering throughout) and G2251 (Fig. 4A, which is published as supporting information on the PNAS web site), respectively (2,4,31). The distinction between the rates of peptide-bond formation by full-length tRNAs and minimal substrates is also consistent with the essential role ...