The ribosome, owing to its exceptional conservation, harbours a remarkable molecular fossil known as the protoribosome. It surrounds the peptidyl transferase center (PTC), responsible for peptide bond formation. While previous studies have demonstrated the PTC activity in RNA alone, our investigation reveals the intricate roles of the ribosomal protein fragments (rPeptides) within the ribosomal core. This research highlights the significance of rPeptides in stability and coacervation of two distinct protoribosomal evolutionary stages. The 617nt 'big' protoribosome model, which associates with rPeptides specifically, exhibits a structurally defined and rigid nature, further stabilized by the peptides. In contrast, the 136nt 'small' model, previously linked to peptidyltransferase activity, displays greater structural flexibility. While this construct interacts with rPeptides with lower specificity, they induce coacervation of the 'small' protoribosome across a wide concentration range, which is concomitantly dependent on the RNA sequence and structure. Moreover, these conditions protect RNA from degradation. This phenomenon suggests a significant evolutionary advantage in the RNA-protein interaction at the early stages of ribosome evolution. The distinct properties of the two protoribosomal stages suggest that rPeptides initially provided compartmentalization and prevented RNA degradation, preceding the emergence of specific RNA-protein interactions crucial for the ribosomal structural integrity.

Department of Cell Biology Faculty of Science Charles University Viničná 7 Prague 12800 Czech Republic

Department of Physical Chemistry University of Chemistry and Technology Technicka 5 16628 Prague Czech Republic

Dipartimento di Biotecnologie Mediche e Medicina Traslazionale Università di Milano Segrate 20054 Italy

Earth Life Science Institute Tokyo Institute of Technology 2 12 1 Ookayama Meguro ku Tokyo 152 8550 Japan

Graduate School of Media and Governance Keio University 5322 Endo Fujisawa 252 0882 Japan

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo n 2 Prague 16610 Czech Republic

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